Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction.

Oncogene addiction is a cellular property by which cancer cells become highly dependent on the expression of oncogenes for their survival. Oncogene addiction can be exploited to design molecularly targeted drugs that kill only cancer cells by inhibiting the specific oncogenes. Genes and cell lines exhibiting oncogene addiction, as well as the mechanisms by which cell death is induced when addicted oncogenes are suppressed, have been extensively studied. However, it is still not fully understood how oncogene addiction is acquired in cancer cells. Here, we take a synthetic biology approach to investigate whether oncogenic mutation or oncogene expression suffices to confer the property of oncogene addiction to cancer cells. We employed human mammary epithelium-derived MCF-10A cells expressing the oncogenic KRAS or BRAF. MCF-10A cells harboring an oncogenic mutation in a single-allele of KRAS or BRAF showed weak transformation activity, but no characteristics of oncogene addiction. MCF-10A cells overexpressing oncogenic KRAS demonstrated the transformation activity, but MCF-10A cells overexpressing oncogenic BRAF did not. Neither cell line exhibited any oncogene addiction properties. These results indicate that the introduction of oncogenic mutation or the overexpression of oncogenes is not sufficient for cells to acquire oncogene addiction, and that oncogene addiction is not associated with transformation activity.

Introduction

Most human cancers develop over a long period of time, from a few years to several decades, as mutations accumulate in various proto-oncogenes and tumor suppressor genes [1, 2]. During this process, cancer cells rewire the intracellular signal transduction system by accumulating mutations and epigenetic changes, and consequently acquire the characteristics of malignant tumors (Fig 1). On the other hand, it is well-established that the overexpression of oncogenes suffices for the neoplastic transformation of non-cancerous cells in vitro and in vivo, resulting in infinite proliferation, anchorage independence, and angiogenesis [3-5]. Therefore, properties that can be acquired over a long period of time appear to be different from the tumorigenesis induced by the proto-oncogenes/tumor suppressor genes activation/inactivation.

Fig 1

Tumorigenic properties and oncogene addiction.

(A) In vivo process by which cancer cells acquire oncogene addiction. (B) The table compares different types of cells with respect to tumorigenic properties and oncogene addiction.

Oncogene addiction (or oncogene pathway addiction) is a characteristic of cancer cells in which malignant cells are dependent for their proliferation and survival on a particular proto-oncogene and/or tumor suppressor gene [6, 7]. Thus, the proliferation and survival of oncogene-addicted cancer cells are dramatically impaired by suppression of the oncogenes. For example, the inhibition of addicted oncogenes with RNAi or small chemical inhibitors causes apoptosis in oncogene-addicted cancer cells, but not in other cells, thereby providing a rationale for molecularly targeted therapy [8]. Imatinib (Gleevec), a BCR-ABL1 kinase inhibitor, and Gefitinib (Iressa), an EGFR inhibitor, are typical examples of drugs successfully targeted to the appropriate molecules and are effective for the treatment of chronic myeloid leukemia (CML) and non-small cell lung cancer, respectively [9]. Several molecular mechanisms by which cancer cells die through acute inhibition of addicting oncogenes selectively required for survival have been reported, including oncogene shock, oncogene amnesia, genetic streaming, synthetic lethality, and others [10, 11]. However, little is known about how and when the property of oncogene addiction is acquired, and which oncogene(s) is prone to cause oncogene addiction, although the phenomenon has been reported to involve epigenetic DNA changes that accompany the development of cancer [12].

The Ras-ERK signaling pathway plays a pivotal role in a wide range of cell functions such as cell proliferation, differentiation, and survival, but also plays a key role in tumorigenesis [13, 14]. Indeed, the KRAS gene is the second-most frequently mutated gene in human cancers, after the p53 gene, and the BRAF gene is also frequently mutated in melanoma and colorectal cancer [2]. KRAS- or BRAF-mutated cancer cells also exhibit oncogene addiction. Suppression of the expression of mutated KRAS by antisense or siRNA caused cell cycle arrest and apoptosis in KRAS-mutated cultured cancer cell lines, and epithelial-mesenchymal transition (EMT) was closely associated with KRAS dependency [15, 16]. Knockdown by RNAi or treatment with a BRAF-selective inhibitor leads to the inhibition of cell proliferation and survival in BRAF-mutated cancer cell lines [17-20]. However, these results were obtained by using cell lines established from human patients, it is impossible to trace when and how oncogene addiction is acquired. Interestingly, the expression of oncogenic HRAS or KRAS has been shown to induce tumor formation in vivo in a doxycycline-dependent manner, and withdrawal of the drug resulted in tumor shrinkage [21, 22]. However, Chin et al. also showed that these cells do not alter their growth rate regardless of doxycycline treatment in vitro [21]. Thus, it remains unclear whether oncogene addiction is achieved by the acquisition of tumorigenic properties through expression of the KRAS or BRAF oncogenes. In this study, we examined whether an oncogenic mutation in a single allele of KRAS or BRAF or overexpression of KRAS or BRAF oncogenes was sufficient to induce oncogene addiction.

Materials and methods

Plasmids, reagents, and antibodies

The plasmids made in this study are listed as follows: pCSIIneo-MCS (multi-cloning site), pCSIIneo-FLAG-BRAF-V600E, pCSIIneo-FLAG-KRAS-G12V, psPAX2 was a gift from Dr. D. Trono, Addgene plasmid #12260) [23]. pCMV-VSV-G-RSV-Rev (a gift of Dr. Miyoshi, RIKEN, Japan). pCSIIbsr-FLAG-BRAF-V600E was a kind gift from Dr. Matsuda (Kyoto University) [24].

The antibodies used for western blot and immunofluorescence analyses are as follows: phospho-anti-Erk1/2 (Thr202/Tyr204) (E10) and anti-Erk1/2 (137F5) were from Cell Signaling Technology; anti-KRAS (clone 3B10-2F2) was from Sigma; anti-Raf-B (F-7) and anti-Tubulin (sc-58886) were from Santa Cruz Biotechnology.

The negative control siPOOL, BRAF targeted siPOOL and KRAS targeted siPOOL were purchased from siTOOLs Biotech.

Cell lines

The A549, H358 (CI-H358), and A375 cell lines were purchased from the American Type Culture Collection. Lenti-X 293T cells were purchased from the Invitrogen. The 293T cells were maintained in DMEM high glucose (Cat#08459–64, Nacalai Tesque) supplemented with 10% fetal bovine serum. H358 cell lines were maintained in RPMI 1640 media (ATCC modification) (Cat#A10491-01, Gibco) supplemented with 10% fetal bovine serum. The A549 and A375 cell lines were maintained in DMEM high glucose (Cat#08459–64, Nacalai Tesque) supplemented with 10% fetal bovine serum and in DMEM high glucose (Cat#08459–64, Nacalai Tesque) supplemented with sodium pyruvate and 10% fetal bovine serum, respectively. All cell lines were maintained at 37°C under 5% CO2.

MCF-10A parental cells, MCF-10A BRAF V600E/+ cells, and MCF-10A KRAS G12V/+ cells (catalog numbers HD PAR-003, HD101-012, and HD101-004) were purchased from Horizon Discovery. KRAS G12V OE and BRAF V600E OE were established through lentivirus-mediated gene transfer into the parental MCF-10A cells. In brief, the lentiviral pCSIIneo or pCSIIbsr vectors were transfected into Lenti-X 293T cells (Clontech) together with the packaging plasmid psPAX2, and pCMV-VSV-G-RSV-Rev by using the linear polyethyleneimine “Max” MW 40,000 (Polyscience). After two days, MCF-10A parental cells were cultured in the virus-containing media in the presence or absence of 8 μg/mL polybrene for 3–4 hrs. Two days after infection, the cells were selected by at least one-week treatment with 150 ug/ml G418 or 10 μg/mL blasticidin (InvivoGen, San Diego, CA). Bulk populations of selected cells were used in this study. An empty vector, pCSIIneo-MCS, was used as a control. All cell lines were maintained at 37°C under 5% CO2 with antibiotics.

MCF-10A cell lines were maintained in the full growth medium, which consisted of DMEM/F12 (1:1) (Cat#11330–032, Gibco) supplemented with 5% horse serum (Cat#16050–122, Invitrogen), 10 mg/ml insulin (Cat#12878–44, Nacalai Tesque), 0.5 mg/ml hydrocortisone (Cat#1H-0888, Invitrogen), 100 ng/ml cholera toxin (Cat#101B, List Biological Laboratories), 20 ng/ml hEGF (Cat#AF-100-15, PeproTech), and 1% penicillin/streptomycin (Cat#26253–84, Nacalai Tesque). For some experiments, partial growth medium and starvation medium were used; the former contained DMEM/F12 (1:1) supplemented with 5% horse serum and 1% penicillin/streptomycin, and the latter consisted of DMEM/F12 (1:1) supplemented with 2% horse serum, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin, and 1% penicillin/streptomycin.

Genomic DNA preparation and sequencing

Genomic DNA was prepared from cells using QuickExtract solution (Nalgene) following the manufacturer’s instruction. PCR amplification was done using KOD FX Neo (Toyobo). PCR primers to amplify DNA were designed to target the oncogene mutated region (15th exon for BRAF and 2nd exon for KRAS). Direct sequencing of PCR products was carried out by FASMAC. The amplification primers were as follows: BRAF-Fw, 5’-ATCTCACCTCATCCTAACACATTTCAAGCCCC-3’; BRAF-Rv, 5’-GACTTTCTAGTAACTCAGCAGCATCTCAGGGCC-3’; KRAS -Fw, 5’-GCCTGCTGAAAATGACTGAA-3’; KRAS-Rv, 5’-AGAATGGTCCTGCACCAGTAA-3’.

Soft-agar colony formation assay

A series of MCF-10A cells (2 x 10^4 per well) were mixed with 0.3% agarose, low gelling temperature (Cat#35640, SIGMA) in the full growth medium, plated on top of a solidified layer of 0.6% agarose in full growth medium [25] in a 6-well plate, and fed every 3 days with full growth medium. Photographs were taken by an OLYMPUS CKX53 inverted microscope with a DP20 digital camera (OLYMPUS). Finally, the colonies were stained with MTT (1 mg/ml in PBS solution) and imaged using an EPSON GT-X900 scanner. The images were analyzed with ImageJ (Fiji), extracting the number of colonies that exceeded a certain threshold intensity. When we combined soft agar assay with the siRNA experiment, 1 nM siRNA-treated cells were embedded in the soft agar, fed only one week later and cultured for 2 weeks. MCF-10A cell lines were transfected with 1 nM siPOOLs, maintained for 2 days in partial growth medium, and then 2 x 10^4 cells were embedded in full growth medium-based soft agar. In the case of cancer-derived cell lines, cells were first transfected with 1 nM siPOOLs in RPMI supplemented with 10% FBS. One day after transfection, the cells were embedded in the soft agar based on RPMI supplemented with 10% FBS. The cell number embedded in the soft agar is as follows: A549, 2 x 10^4 cells; H358, 4 x 10^4 cells; A375, 1 x 10^4 cells.

Western blot analysis

Cells were washed once with PBS and lysed directly in 1x SDS sample buffer (1 M Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.2% bromophenol blue, and 10% 2-mercaptoethanol). When the number of cells decreased due to knock-down of KRAS or BRAF, the volume of SDS sample buffer was reduced for cell lysis. After sonication and heat denaturation by boiling, the samples were separated by premade 5–20% gradient SDS-polyacrylamide gel electrophoresis (PAGE) (Nakalai or Atto) and transferred to Immobilon-FL Polyvinylidene Difluoride (PVDF) membranes (Millipore, Billerica, MA). After blocking with skim milk (Morinaga, Tokyo) or Odyssey blocking buffer (LI-COR), the membranes were incubated with primary antibodies diluted in skim milk, BSA, or Odyssey blocking buffer (LI-COR), followed by secondary antibodies diluted in Odyssey blocking buffer. Proteins were then detected by an Odyssey Infrared scanner (LI-COR) and analyzed by using the Odyssey software. The detection conditions are as follows: Resolution, 168 μm (Figs 2F and 5F KRAS and BRAF, and S3C, S4A and S5C Figs) or 84 μm (Fig 5F empty vector, and S1D, S2A, S2C, S3A and S5A Figs); sensitivity (scanning speed), normal (Figs 2F and 5F KRAS and BRAF, and S3C, S4A and S5C Figs) or low (Fig 5F empty vector, and S1D, S2A, S2C, S3A and S5A Figs). For the analysis of ERK phosphorylation in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines, cells were seeded with full growth medium for 1 day before the serum starvation; washed once with PBS and changed to starvation medium for 24 hrs; and finally treated with 10 ng/ml EGF for 10 min. In the case of ERK phosphorylation analysis in MCF-10A cells overexpressing KRAS G12V or BRAF V600E, the MCF-10A cells were seeded with full growth medium or starvation medium, cultured for 24 hours, and treated with 10 ng/ml EGF for 10 min. For the quantification, the intensity of ERK1/2 or Tubulin signal was used as a loading control.

Fig 2

Characterization of MCF-10A cells harboring a single allele mutation of KRAS G12V or BRAF V600E.

(A and B) Morphology of the indicated MCF-10A cells seeded onto a cell culture dish for two days (A) or seeded in soft agar for seven days (B). (C) Morphology of the indicated MCF-10A cells seeded in soft agar for 4 weeks. (D) Representative images of MTT-stained colonies of the indicated MCF-10A cells seeded in soft agar for 4 weeks. (E) The mean number of colonies (left) and the mean colony size (μm2) (right) are shown with the SD. Dots indicate actual data points. The numbers of experiments are as follows: Parental, n = 4 (left) and 3 (right); KRAS G12V/+, n = 3 and 4; BRAF V600E/+, n = 3 and 4. (F) Western blot analysis from the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines under the indicated conditions. (G) Quantification of ERK phosphorylation in panel F. Relative pERK/ERK values normalized by parental MCF-10A cells cultured in full growth medium condition are shown with the SD (n = 3). Dots indicate actual data points. The results of statistical analysis are shown in S1B Fig. * p < 0.05, ** p < 0.01.

Fig 5

Characterization of MCF-10A cells overexpressing KRAS G12V or BRAF V600E.

(A and B) Morphology of the indicated MCF-10A cells seeded onto a cell culture dish for two days (A) or seeded in soft agar for seven days (B). (C) Morphology of the indicated MCF-10A cells seeded in soft agar for 2 weeks. A white asterisk indicates the ruptured spheroid in KRAS G12V OE. (D) Representative images of MTT-stained colonies of the indicated MCF-10A cells seeded in soft agar for 4 weeks. (E) The mean number of colonies (left) and the mean colony size (μm2)(right) are shown with the SD. Dots indicate actual data points. The numbers of experiments are as follows: Empty vector, n = 4; KRAS G12V OE, n = 4; BRAF V600E OE, n = 3. (F) Western blot analysis of the parental MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines under the indicated conditions. (G) Quantification of ERK phosphorylation in panel F. Relative pERK/ERK values normalized by empty vector (left) and parental cells (right) under the full growth medium condition are shown with the SD (empty vector, n = 4; KRAS G12V OE, n = 5; BRAF V600E OE late, n = 5). Dots indicate actual data points. The results of statistical analysis are shown in S4B Fig. p < 0.05, ** p < 0.01.

Crystal violet staining

For siRNA-mediated knockdown experiments to examine oncogene-addiction, we used siPOOLs. Reverse transfection of cancer cell lines and MCF-10A cell lines with siPOOLs was performed in Nunc edge 2.0 (96-Well Plates, Thermo Fisher) using Lipofectamine RNAiMax reagent (Invitrogen) according to the siPOOLs transfection protocol. Seeding density and siPOOL concentrations were modified depending on the cell lines and culture conditions used. After 3 to 4 days of siPOOL treatment, cells were washed once with PBS and stained with 0.1% crystal violet for 10 min, then washed three times with PBS and photographed with an EPSON GT-X900 scanner (1200 dpi). Relative cell number calculations were performed by ImageJ (Fiji) in comparison with the corresponding cell lines treated with a negative control siPOOL. For cancer cell lines, 1 nM siPOOL-reverse transfected cells were cultured in the RPMI 1640 medium (ATCC modification) (Cat#A10491-01, Gibco) supplemented with 10% fetal bovine serum for 3–4 days with the following cell densities, 6x10^3 cells/well for H358, 1x10^3 cells/well for A549, and 1-2x10^3 cells/well for A375. And knock-down experiment of the derivative cell lines of MCF-10A was performed the following conditions; in partial growth medium condition, reverse transfection in 6x10^3 cells with 1 nM siPOOLs for 3–4 days, and in starvation medium condition, reverse transfection in 3x10^3 cells with 0.5 nM siPOOLs for 3–4 days.

Statistical analysis

In Figs 1–6 and S1 Fig, the Kruskal−Wallis test, a non-parametric alternative to the one-way ANOVA, was adopted, because the data points are non-normally distributed. When p-value of the Kruskal-Wallis test was less than 0.05, as a post-hoc analysis the Conover−Iman test with the Bonferroni−Holm Correction for multiple testing was adopted to calculate p-values. These statistical tests were conducted on Python 3 and SciPy. In S2, S3 and S5 Figs, a paired t-test was adopted with the Microsoft excel software.

Fig 6

Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells overexpressing KRAS G12V or BRAF V600E.

(A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in parental MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines grown in the partial growth medium. Three days after transfection with siRNA, relative cell densities were determined by crystal violet staining. (A) Representative 96-well plates are shown. (B) The mean relative cell number is shown with the SD. The numbers of experiments are as follows: Parental, n = 4; KRAS G12V OE, n = 4; BRAF V600E OE late, n = 4. Dots indicate actual data points. (C) Cell growth assays in empty vector-introduced MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines grown in the starvation medium for four days with the indicated siRNA. Relative cell densities were determined by crystal violet staining. (lower) Representative 96-well plates are shown. (upper) Mean relative cell numbers are shown with the SD (empty vector control, n = 9; KRAS G12V OE, n = 25; BRAFV600E OE late, n = 11). Dots indicate actual data points. (D and E) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the KRAS G12V OE cell line grown in the starvation medium. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. (D) Representative 96-well plates are shown. (E) The mean relative cell number is shown with the SD (Control, n = 25; KRAS, n = 24; BRAF, n = 24). Dots indicate actual data points. (F) Representative images of MTT-stained colonies of the indicated cells seeded in soft agar for 2 weeks. (G) The mean number of colonies (left) and the mean colony size (μm2)(lower) are shown with the SD (KRAS G12V OE, n = 6). Dots indicate actual data points. * p < 0.05, ** p < 0.01.

Results

In vitro characterization of MCF-10A cells harboring a single allele mutation of KRAS G12V or BRAF V600E

The human normal mammary gland-derived MCF-10A cell lines were used in this study. This is because MCF-10A was spontaneously immortalized without defined factors [26], the cell line is not tumorigenic, i.e., they are not able to grow under anchorage-independent conditions or to form tumors when injected subcutaneously into nude mice [27], it does not have mutations in KRAS and BRAF, and it is easy to culture. In addition, it is beneficial to use MCF-10A because of the availability of cell lines with KRAS G12V and BRAF V600E as we mention below. On the other hand, it has been reported that MCF10A lacks a tumor suppressor gene, p16, which may render the cell line immortalized. Therefore, it should be noted that it is substantially different from normal mammary epithelial cells (see Discussion).

To reconstitute oncogene addiction, we first obtained MCF-10A cells harboring KRAS G12V or BRAF V600E mutation, which were generated by genome editing with adeno-associated virus (hereafter referred to as KRAS G12V/+ or BRAF V600E/+ cells) [27, 28]. Mutation of a single allele of KRAS G12V and BRAF V600E was confirmed by direct sequencing (S1A Fig). KRAS G12V/+ cells grew as efficiently as parental MCF-10A cells onto two-dimensional dishes, showing islands of densely packed cells (Fig 2A). Meanwhile, BRAF V600E/+ cells exhibited more scattered and fibroblastic morphology (Fig 2A). We next evaluated anchorage-independent colony formation in soft agar, which is a feature of transformation [29]. Seven days after seeding in the soft agar, the parental MCF-10A cells hardly proliferated, whereas KRAS G12V/+ cells grew slowly and formed small spheroids (Fig 2B). This result is not consistent with the previous report using the same cells [28], possibly because of the difference of the experimental conditions under which the seeding cell number (3x10^4 cells vs 2x10^4 cells), the top layer agar concentration (0.3% vs 0.4%), the interval of medium addition (every 3 days vs every 1 week), and/or incubation time (4 weeks vs 3 weeks). Under the same condition, BRAF V600E/+ cells formed larger and more spheroids than KRAS G12V/+ cells (Fig 2B) in good agreement with the previous report [27], suggesting an increase in transformation activity in BRAF V600E/+ cells. Despite the smaller size of colonies, the number of colonies in KRAS G12V/+ cells was comparable to that of BRAF V600E/+ cells 4 weeks after seeding in the soft agar (Fig 2C–2E). Consistent with these data, BRAF V600E/+ cells showed significantly higher basal phosphorylation of ERK, downstream of KRAS and BRAF, under normal and serum-starved conditions (Fig 2F and 2G, S1B Fig). Parental and KRAS G12V/+ cells responded well to EGF stimulation, while BRAF V600E/+ cells demonstrated less sensitivity to EGF, probably because of its higher basal activity (Fig 2F and 2G, S1B Fig). The expression levels of KRAS and BRAF showed no substantial changes in these cell lines (S1C–S1E Fig), suggesting that the differences between parental cells and the mutant cell lines were attributable to the increased activity of KRAS and BRAF. These results indicated that a single allele mutation of BRAF V600E in MCF-10A cells enhances transformation activity in culture, while a single allele KRAS G12V results in cells with similar or slightly increased in vitro transformation activity.

Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells harboring a single allele mutation of KRAS G12V or BRAF V600E

We next quantified to what extent cells were addicted by the expression of KRAS or BRAF oncogene. For this purpose, the effect of KRAS or BRAF ablation on cell growth was examined with crystal violet staining and RNA interference (RNAi) [16, 30]. In addition, to reduce the off-target effect of RNAi, siPOOLs were used to deplete KRAS and BRAF; siPOOLs dilute the off target effects by pooling multiple siRNAs against the target genes [31]. The siPOOLs for KRAS or BRAF do not discriminate between wild-type and mutant sequences of KRAS or BRAF.

As a control, we used two lung cancer-derived cell lines, A549 (homozygous KRAS G12S mutation; KRAS non-addicted) and H358 (heterozygous KRAS G12C mutation; KRAS-addicted) [16], and melanoma-derived cell line, A375 (homozygous BRAF V600E mutation; BRAF-addicted) [19]. The knockdown of KRAS or BRAF with siPOOLs was confirmed in these cell lines (S2A and S2B Fig). A549 cells showed modest inhibition of cell growth by KRAS depletion (Fig 3A and 3B). H358 and A375 cells exhibited strong suppression of cell growth by KRAS and BRAF ablation with the siPOOLs (siRNA), respectively (Fig 3A and 3B). In A375 cells, knock-down of the BRAF gene caused a concomitant decrease in both pERK and cell proliferation, which was consistent with previous reports showing that cancer cells harboring BRAF V600E are addicted to ERK pathway (S2A and S2B Fig) [32]. In H358 cells cultured in RPMI supplemented with 10% FBS, knock-down of the KRAS gene suppressed cell proliferation but not pERK with statistical significance (S2A and S2B Fig). Meanwhile, we could reproduce that ERK phosphorylation was significantly reduced in H358 cells cultured in 5% FBS medium as previously shown (S2C and S2D Fig) [16]. Importantly, the inhibition of cell growth was observed even under a full growth condition for these cancer cell lines; these cells were indeed addicted to the expression of the KRAS or BRAF oncogene for their cell growth.

Fig 3

Evaluation of oncogenic KRAS or BRAF addiction in cancer-derived cell lines.

(A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the A549, H358, and A375 cell lines. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. (A) Representative 96-well plates are shown. (B) The mean relative cell number is shown with the SD. Dots indicate actual data points. The numbers of experiments are as follows: A549, n = 6; H358, n = 6; A375, n = 6. (C) Representative images of MTT-stained colonies of the indicated cells seeded in soft agar for 2 weeks. (D) The mean number of colonies (upper) and the mean colony size (μm2)(lower) are shown with the SD. Dots indicate actual data points. The numbers of experiments are as follows: A549, n = 6; H358, n = 6; A375, n = 6. * p < 0.05, ** p < 0.01.

We further investigated the effect of KRAS or BRAF knock-down on anchorage independent growth in these cancer-derived cell lines. The cells were introduced with siPOOLs and embedded in soft agar 1 day after the siPOOLs transfection. Of note, the effect of siPOOLs-mediated knock-down would not last for a long time because of the dilution of siRNA through cell proliferation, and therefore we presume that siPOOLs-mediated knock-down affects cell survival and proliferation in the early stages of the soft agar colony formation. Nevertheless, we found that the number of colonies in A549 cells and H358 cells was significantly reduced by KRAS depletion (Fig 3C and 3D). BRAF depletion decreased the number of colonies in A375 cells and H358 cells (Fig 3C and 3D). These results indicate the involvement of oncogenic KRAS or BRAF in in vitro transformation properties.

We then examined whether KRAS G12V/+ and BRAF V600E/+ cells were addicted to their oncogenes. If oncogene addiction has been acquired, we expect that knock-down of the oncogene would result in phenotypes such as apoptosis in oncogene addicted cells, but not in normal cells and oncogene non-addicted cells. First, siPOOLs were introduced into parental, KRAS G12V/+, and BRAF V600E/+ MCF-10A cells cultured in “full growth medium”. However, we were unable to reproducibly quantify the effect of RNAi on KRAS or BRAF ablation and cell growth. This is because the seeding density at the time of cell passage has a great impact on the assay even a few days after the passage. Therefore, we used “partial growth medium”, which contained DMEM and serum without the supplements included in the “full growth medium”. Under the culture condition with the partial growth medium, MCF-10A cells grew and proliferated slowly, making it easier to assess the depletion of KRAS and BRAF (S3A and S3B Fig). Parental MCF-10A cells did not show substantial change in cell growth by the knock-down of the KRAS or BRAF gene (Fig 4A and 4B). We found that neither KRAS nor BRAF depletion resulted in substantial effect on cell growth in KRAS G12V/+ and BRAF V600E/+ cells (Fig 4A and 4B), indicating that cell growth in these cells was not dependent on the expression of the KRAS or BRAF oncogene.

Fig 4

Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells harboring a single allele mutation of KRAS G12V or BRAF V600E.

(A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines grown in the partial growth medium. Three days after transfection with siRNA, relative cell densities were determined by crystal violet staining. (A) Representative 96-well plates are shown. (B) The mean relative cell number is shown with the SD (Parental, n = 4; KRAS G12V/+, n = 4; BRAF V600E/+, n = 5). Dots indicate actual data points. (C) Cell growth assays in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines grown in starvation medium for four days with the indicated siRNA. Relative cell densities were determined by crystal violet staining. (Lower) Representative 96-well plates are shown. (Upper) Mean relative cell numbers are shown with the SD (Parental, n = 9; KRAS G12V/+, n = 22; BRAF V600E/+, n = 25). Dots indicate actual data points. (D and E) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the KRAS G12V/+ and BRAF V600E/+ cell lines grown in starvation medium. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. (D) Representative 96-well plates are shown. (E) The mean relative cell number is shown with the SD (KRAS G12V/+, n = 25; BRAF V600E/+, n = 25). Dots indicate actual data points. (F) Representative images of MTT-stained colonies of the indicated cells seeded in soft agar for 2 weeks. (G) The mean number of colonies (left) and the mean colony size (μm2) (lower) are shown with the SD. Dots indicate actual data points. The numbers of experiments are as follows: KRAS G12V/+, n = 6; BRAF V600E/+, n = 6. * p < 0.05, ** p < 0.01.

The oncogenic mutation in a single allele of KRAS or BRAF leads to sustained proliferative signaling [2]. Indeed, the introduction of oncogenic mutation in KRAS or BRAF enabled MCF-10A cells to grow in the culture medium without EGF and serum, namely, the “starvation medium” (Fig 4C). We confirmed depletion of KRAS and BRAF under the starvation condition (S3C and S3D Fig). Interestingly, the growth factor independence was derived from KRAS expression in KRAS G12V/+ cells, whereas it was not derived from BRAF expression in BRAF V600E/+ cells (Fig 4D and 4E). We then investigated the involvement of oncogenic mutation of KRAS or BRAF in anchorage-independent growth in KRAS G12V/+ and BRAF V600E/+ cells. KRAS depletion highly reduced the number of colonies in these two cell lines (Fig 4F and 4G). Knock-down of the BRAF gene also attenuated the number of colonies in these cell lines, but the effect was stronger in BRAF V600E/+ cells than in KRAS G12V/+ cells (Fig 4F and 4G). Although these results indicate that oncogenic mutation in KRAS or BRAF is involved in the acquisition of the ability for growth factor-independent and anchorage-independent growth, they are not suitable for evaluation of oncogene addiction because parental cells did not proliferate in the starvation medium and in the soft agar. In sum, we concluded that KRAS G12V/+ cells and BRAF V600E/+ cells were not addicted to the oncogene, even though these cells acquired the ability of growth factor-independent and anchorage-independent growth.

In vitro characterization of MCF-10A cells overexpressing KRAS G12V or BRAF V600E

Next, we examined whether overexpression of KRAS G12V or BRAF V600E induced the property of oncogene addiction. The FLAG-tagged KRAS G12V or BRAF V600E oncogene was introduced into parental MCF-10A cells through lentivirus, producing MCF-10A cells over-expressing KRAS G12V or BRAF V600E (hereinafter referred to as “KRAS G12V OE” or “BRAF V600E OE” cells). As a control, we introduced the empty vector into MCF-10A cells through lentivirus (referred to as “empty vector”). The expression levels of KRAS, BRAF, and ERK were compared with parental cells (S1C–S1E Fig). During the course of experiments, we recognized that long-term culture of BRAF V600E OE cells reduced the expression levels of BRAF V600E and ERK phosphorylation levels (S4A Fig), probably due to the adaptation by a reduction of BRAF V600E expression through gene silencing and/or negative feedback mechanisms [33]. Thus, we referred to early-passage (< 1 week from the establishment of cell lines) and late-passage (> 1 week) cells as BRAF V600E OE early cells and BRAF V600E OE late cells, respectively.

The morphology of KRAS G12V OE cells cultured on the plastic dish was scattered and fibroblastic, whereas BRAF V600E OE late cells exhibited a typical epithelial cell shape to the same extent that the empty vector-introduced control cells did (Fig 5A). KRAS G12V OE cells displayed rapid cell growth in soft agar, forming large colonies one week after seeding (Fig 5B). Raptured spheroids were observed two weeks after seeding (Fig 5C), and finally, many small colonies were observed four weeks after seeding (Fig 5D and 5E). BRAF V600E OE late cells showed no anchorage-independent growth in soft agar, but BRAF V600E OE early cells formed small colonies in soft agar after one week (Fig 5B), large colonies at two weeks that were comparable in size to those in anchorage-independent growth of BRAF V600E/+ cells for 4-weeks (S4C Fig), and finally, a small number of larger colonies at 4 weeks after seeding (Fig 5D and 5E). Consistent with the change in cell morphology and anchorage-independent growth, strong ERK phosphorylation was maintained in KRAS G12V OE cells under all conditions (Fig 5F and 5G). Despite the higher basal ERK phosphorylation in BRAF V600E OE early cells (S4A Fig), BRAF V600E OE late cells showed comparable or slightly higher levels of ERK phosphorylation in comparison to control cells (Fig 5F and 5G).

Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells overexpressing KRAS G12V or BRAF V600E

Finally, we quantified the effect of depletion of KRAS and BRAF with siPOOLs (siRNA) on cell growth in KRAS G12V OE and BRAF V600E OE late cells, respectively. The knockdown efficiency of siPOOLs targeting KRAS and BRAF was confirmed by western blotting (S5A and S5B Fig). To our surprise, KRAS or BRAF ablation only slightly reduced the cell growth rate in KRAS G12V OE or BRAF V600E OE late cells cultured in partial growth medium (Fig 6A and 6B). Of note, KRAS G12V OE cells have a spindle-like morphology (Fig 5A) that results in weaker crystal violet staining compared to other cells, but does not alter the rate of cell growth.

Like KRAS G12V/+ cells, KRAS G12V OE cells demonstrated growth factor independence (Fig 6C), and this was dependent on the expression of KRAS (Fig 6D and 6E, S5C and S5D Fig). BRAF V600E OE late cells showed a modest increase in cell growth under the serum starvation condition (Fig 6C). In addition, anchorage-independent growth in KRAS G12V OE cells were attenuated by KRAS depletion (Fig 6F and 6G). Taken together, the overexpression of KRAS G12V or BRAF V600E enhanced more or less in vitro transformation activity, but it did not suffice to induce oncogene addiction in MCF-10A cells.

Discussion

In this study, we examined the association between oncogene addiction and in vitro tumorigenic properties (Fig 7A). In the human normal mammary gland-derived MCF-10A cell lines, an oncogenic mutation in a single allele of the KRAS or BRAF gene induced modest anchorage-independence, proliferative capacity, and phosphorylation of ERK, while the cells did not exhibit oncogene addiction. Similarly, MCF-10A cells overexpressing the oncogenic KRAS G12V or BRAF V600E protein demonstrated several properties of in vitro transformation properties, but did not show any signs of oncogene addiction. From these results, we conclude that, at least in the KRAS or BRAF gene of MCF-10A, the introduction of an oncogenic mutation or overexpression of an oncogene does not ensure the acquisition of oncogene addiction, and the properties of in vitro transformation are not necessarily coupled with oncogene addiction.

Fig 7

Model for the acquisition of oncogene addiction.

(A) A table summarizing the results of this study. (B) Schematic diagram showing how oncogene addiction is acquired.

Why was the oncogenic mutation or over-expression of KRAS or BRAF in MCF-10A cells not sufficient to induce oncogene addiction? A reasonable possibility is that, after the introduction of the first oncogene mutation in vivo, tumor cells accumulate the oncogenes and/or tumor suppressor gene mutations with epigenetic alterations over a long period of time, gradually acquiring oncogene addiction (Fig 7B). Indeed, sensitivity to BRAF and MEK inhibitors, a feature of BRAF addiction, has been associated with distinct phenotype plasticity of the differentiation state and global alterations in gene expression programs in BRAF-mutated melanomas [34-36]. Further, it has been reported that a gene expression pattern associated with epithelial-mesenchymal transition (EMT) correlates with KRAS addiction [16]. Malignant tumor cells exhibiting the property of oncogene addiction may undergo environmental changes in vivo that render the cells oncogene-addicted. In other words, the in vitro culture of MCF-10A cells may be insufficient to cause genetic and/or epigenetic alteration leading to oncogene addiction. It would be of critical importance to identify such an environment or stimuli in order to enhance the effects of molecularly targeted drugs. Another (and not mutually exclusive) possibility is that oncogene addiction may be tissue-specific; thus mammary gland-derived MCF-10A cells may be inherently incapable of acquiring KRAS or BRAF addiction. Oncogenic KRAS or BRAF addiction has been found in lung and colon cancers and melanomas, where KRAS or BRAF is frequently mutated [16]. Human breast cancers rarely show KRAS and BRAF mutations, whereas mutations in genes that activate the PI3K-Akt pathway have been reported [37]. Interestingly, Her2 amplification and PIK3CAH1047R-positive breast cancer exhibited PI3K dependency in a mouse model [38], suggesting that genes that are strongly involved in tumorigenesis in a specific tissue are also likely to show oncogene addiction.

In this study, we observed phenotypic differences in MCF-10A cells expressing oncogenic KRAS or BRAF. These differences may have been due to differences in the expression levels and/or differences between KRAS G12V and BRAF V600E. Significant increases in ERK phosphorylation and tumorigenic activity were not observed in KRAS G12V/+ cells (Fig 2), whereas both changes were observed in KRAS G12V OE cells, which expressed oncogenic KRAS G12V at approximately 20-fold the level of endogenous KRAS (S1C and S1D Fig). Interestingly, neither case demonstrated an oncogene-induced senescence phenotype. It has been reported that cellular senescence is induced when the expression of HRAS G12V is twice that of endogenous RAS in MCF-10A cells and human mammary epithelial cells/hTERT [39]. The higher expression of KRAS G12V in mouse mammary epithelium in vivo has been found to induce cellular senescence in the p53-p16 pathway dependent manner [40]. However, homozygous loss of p16 in MCF-10A cells has been demonstrated [41], which probably makes the cells immortal and resistant to cellular senescence caused by KRAS G12V overexpression. These results strongly suggest a dose-dependent switch between cellular senescence and tumorigenesis of KRAS G12V in mammary epithelial cells. With respect to BRAF, our results showed that the expression of BRAF V600E from the endogenous gene locus enhanced ERK phosphorylation and in vitro transformation activity (Fig 2), but these characteristic features were gradually diminished when BRAF V600E was overexpressed, and eventually, cells with low BRAF V600E expression were selected (Fig 5 and S4 Fig). Although BRAF V600E-induced cellular senescence has not been reported in mammary epithelial cells, melanocytes and fibroblasts have been reported to show cellular senescence by overexpression of BRAF V600E [42]. It is reasonable to assume that overexpression of BRAF V600E induced cellular senescence, thereby leading to the selection of cells with low BRAF V600E expression. However, the susceptibility of oncogene-induced senescence is cell type-dependent. In fibroblasts, overexpression of oncogenic KRAS induces senescence [43], while endogenous KRAS G12D expression enhances cell proliferation [44]. In the future, a more quantitative investigation will be needed to reveal the relationship between the expression level of oncogenes and the consequence of tumorigenesis and cellular senescence.

Future studies should focus on the development of an experimental system for the acquisition of oncogene addiction in various cell lines including lung and pancreas-derived cells. It has been reported that overexpression of Myc followed by suppression leads to apoptosis as a model of oncogene addiction [45]. However, it is technically difficult to trace the process by which cells acquire oncogene addiction in an in vivo model. Therefore, it will be necessary to develop an in vitro experimental system for the acquisition of oncogene addiction. Understanding what environmental changes lead to oncogene addiction and what state changes the cell undergoes in the process will be important in augmenting the effects of molecularly targeted drugs.

Characterization of MCF-10A cells harboring an oncogenic mutation in a single allele of KRAS or BRAF.

(A) Genome sequence of KRAS gene (left) and BRAF gene (right) in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines. (B) The results of statistical analysis in Fig 2G are shown in the heatmap. See Materials and Methods for the details of the statistical analysis. (C-E) Expression levels of KRAS, BRAF, and ERK in MCF-10A cell lines used in this study. (C) Western blot analysis of the MCF-10A cell lines. (D) Quantification of the expression levels of KRAS, BRAF, and ERK in panel C. Relative values normalized by parental MCF-10A cells cultured in full growth medium condition are shown with the SD (n = 3 in all cell lines). Dots indicate actual data points. (E) The results of statistical analysis are shown in the heatmap. * p < 0.05, ** p < 0.01.

(EPS)

Click here for additional data file.

Knock-down efficiencies in cancer-derived cell lines.

(A and B) Knock-down efficiencies of KRAS and BRAF using targeted siPOOLs in A549 cells, H358 cells, and A375 cells cultured in RPMI supplemented with 10% FBS were analyzed by western blotting. (A) The representative western blot images are shown. (B) Quantification of the expression levels of KRAS, BRAF, ERK, and pERK in panel A. Relative values normalized by the value of negative control are shown with the SD. The numbers of experiments are as follows: A549, n = 5; H358, n = 5; A375, n = 6. Dots indicate actual data points. (C and D) Knock-down efficiencies of KRAS and BRAF using targeted siPOOLs in H358 cells cultured in RPMI supplemented with 5% FBS were analyzed by western blotting. (C) The representative western blot images. (D) Quantification of the expression levels of KRAS, BRAF, ERK, and pERK in panel C. Relative values normalized by the value of negative control are shown with the SD (n = 4). Dots indicate actual data points. * p < 0.05, ** p < 0.01.

(EPS)

Click here for additional data file.

Knock-down efficiencies in MCF-10A cells harboring an oncogenic mutation in a single allele of KRAS or BRAF.

(A and B) Knock-down efficiencies of KRAS and BRAF using targeted siPOOLs in the indicated MCF-10A cells cultured in the partial growth medium were analyzed by western blotting. (A) The representative western blot images are shown. (B) Quantification of the expression levels of KRAS, BRAF, ERK, and pERK in panel A. Relative values normalized by the value of negative control are shown with the SD. The numbers of experiments are as follows: Parental, n = 3; KRAS G12V/+, n = 3; BRAF V600E/+, n = 3. Dots indicate actual data points. (C and D) Knock-down efficiencies of KRAS and BRAF using targeted siPOOLs in the indicated MCF-10A cells cultured in the starvation medium were analyzed by western blotting. (C) The representative western blot images are shown. (D) Quantification of the expression levels of KRAS, BRAF, ERK, and pERK in panel C. Relative values normalized by the value of negative control are shown with the SD. The numbers of experiments are as follows: KRAS G12V/+, n = 4; BRAF V600E/+, n = 3. Dots indicate actual data points. * p < 0.05, ** p < 0.01.

(EPS)

Click here for additional data file.

(A) BRAF and pERK levels in the indicated MCF-10A cell lines were analyzed by western blotting. The representative images are shown. (B) The results of statistical analysis in Fig 5G are shown in the heatmap. * p < 0.05, ** p < 0.01.

(EPS)

Click here for additional data file.

Knockdown efficiencies in MCF-10A cells overexpressing KRAS G12V or BRAF V600E.

(A and B) Knock-down efficiencies of KRAS and BRAF using targeted siPOOLs in the indicated MCF-10A cells cultured in the partial growth medium were analyzed by western blotting. (A) The representative western blot images are shown. (B) Quantification of the expression levels of KRAS, BRAF, ERK, and pERK in panel A. Relative values normalized by the value of negative control are shown with the SD. The numbers of experiments are as follows: Empty vector, n = 3; KRAS G12V OE, n = 3; BRAF V600E OE late, n = 3. Dots indicate actual data points. (C and D) Knock-down efficiencies of KRAS and BRAF using targeted siPOOLs in KRAS G12V OE cells cultured in the starvation medium were analyzed by western blotting. (C) The representative western blot images are shown. (D) Quantification of the expression levels of KRAS, BRAF, ERK, and pERK in panel C. Relative values normalized by the value of negative control are shown with the SD (n = 3). Dots indicate actual data points. * p < 0.05, ** p < 0.01.

(EPS)

Click here for additional data file.

(PDF)

Click here for additional data file.

30 Oct 2020

PONE-D-20-30780

Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction.

PLOS ONE

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Reviewer #2: N/A

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Reviewer #1: Here Ito and colleagues have submitted a manuscript to explore when cells become ‘oncogene addicted’. They use modified MCF10A cells as a surrogate for normal mammary epithelial cells expressing physiologic levels or elevated levels of oncogenic versions of KRAS (G12V) or BRAF (V600E). Physiologic levels were obtained via knock-in mutations and overexpression was attained through lentiviral transduction. They assessed cell number reduction as a surrogate for transformation caused by knockdown of the oncogene in question.

Concerns:

1. Assessment of oncogenicity:

For a tumour or cell line to be oncogenically addicted it must be shown to be oncogenic and that the removal of the oncogene then prevents oncogenicity. (a) the authors never show that these cells are capable of forming tumours in recipient animals. While soft agar assay positivity does correlate with tumor formation this is not always the case. (b) The bigger concern however, is that there is no use of soft agar assays (ie transformation assay) to determine whether the cells remain transformed after knockdown of the oncogene in question. This undoubtedly is because siRNA pools were used, which while being a standard first start to addressing a requirement, fail in any longer-term assessment of requirement. Longer term experiments can be accomplished through the use of lentiviral/retroviral vectors (even with inducible knockdown) that are completely compatible with longer term assays and make the cell population more uniform. Additionally, for BRAF the use of BRAFV600E specific drugs could have been employed in longer assays. Both approaches would work for tumorgenicity assays and soft agar transformation assays (if the authors do not have access to resources necessary for the mouse work). Without these, the only thing that the authors are able to study is a deviation in cell number and as proliferation in culture is not reflective of transformation or oncogenicity this makes statements about addiction difficult to assess. For example, while it is true that a severe reduction in proliferation would most likely cause a decrease in tumorigenicity, many proliferating cells are not themselves transformed - for instance the parental MCF10A cells are proliferative but not tumorigenic. Thus, if one uses proliferation as the sole assay for oncogene addiction then you are likely to mischaracterize phenotypes. Since the authors haven’t measured transformation as a readout then they really cannot say whether or not their cells are oncogene addicted. I encourage the authors to either assess this using lentiviral knockdown or alter the text considerably to reflect what they are assessing here (growth factor independence effects on proliferation).

2. Choice of cells for this study:

This reviewer is slightly concerned about the use of MCF10a cells as a surrogate of normalcy. Despite being widely used, MCF10A cells do have properties that distinguish them from primary mammary epithelial cells (immortality as well as genomic rearrangements Cancer Genet Cytogenet, 2005 Nov;163(1):23-9.). But this concern is eclipse by the concern that the authors chose to study the effects of KRAS/BRAF mutations in breast epithelial cells. A quick search of the TCGA reveals that in ~6800 breast tumors 62 patients had 62 mutations in one or both of these genes (ie approximately 1% of breast cancer samples). One wonders why there is a lack of RAS/RAF mutations. Are mammary cells simply resistant to hyperactivation of the RAS/RAF? Are they sensitive to such signals and unduce senescence? Indeed, there is data to support both notions (Nature Cell Biol volume 9,pages493–505 (2007)). The authors should state why they used this particular cell line for their studies in the text and in the discussion, what the caveats are for using such lines (vs normal mammary epithelial cells)

3. Data quality, presentation analysis:

a) The supplemental data supporting knockdown in the cancer derived lines and MCF10A-derivatives was rather weak. These are not very convincing blots (specifically those in Supp fig S2/S3). It is unclear how many times these were done. Quantification would be bolstered by repetition and statistics. In particular a direct comparison of MCF10A-derivatives should be presented side-by-side on one blot.

b) Are the blots within a given panel at the same levels of exposure? For example, figure 1D shows quantification relative to parental controls in full growth media but the blots are shown as being separate. In other situations (FgiS3a) the blots are exposed differently.

c) Figures involving protein quantification (2d, 4D) and cell number (3b,d,e,g5bce) should display that actual data points in addition to a graph with error bars) AND indicate statistical significance where present (with a statement or indicator where it is not). This would allow the reader to more accurately assess the data presented.

This reviewer was surprised by the statement that the MCF10A-derived cells grew too fast to “reproducibly quantify the effect of RNAi on KRAS or BRAF ablation”. Do these cells grow faster than A549s or the other tumor derived cell lines? If not, the issue would most likely be due to transfection efficiency.

4. Finally this reviewer is concerned about inconsistencies with the published literature. MCF10A cells with KRAS G12D knockin mutations have different properties than the cells used here (the authors point this out). Are these cells different or is the same source? (the ones used in this paper are purchased) If they are the same cells, then why would there be a difference? If they are different cells, then what could be an explanation that could reconcile these data? The use of siRNAs to KRAS do not replicate data seen by others (including Jeff Settelman’s group). Again, this is perplexing and without explanation.

5. Minor Text issues:

Line 134 reads, “…mutation of BRAF V600E in MCF-10A cells enhances in vitro tumorigenic activity… ”. The authors do not measure tumor formation, they are measuring transformation in culture. The text should be revised.

Figure 6 uses the word “tumorigenisities ”. I am not sure this is a word.

Reviewer #2: This paper by Ito et el. uses cell line systems to investigate the induction of oncogene addiction following endogenous or exogenous expression of mutant KRAS(G12V) or BRAF(V600E). The authors conclude that expression of single mutant oncogenes in defined in vitro cell culture models does not lead to oncogene addiction. Unfortunately, the data as presented do not support the major conclusion of the paper. Firstly, as the authors acknowledge (lines 250-251), KRAS or BRAF mutations are rarely found in breast cancer. Therefore, the use of MCF10A mammary epithelial cells as the only model for the experiments limits the ability to generalize conclusions. Additional cell line models should be tested using lung and pancreas-derived cell lines. Secondly, the authors constantly refer to “tumorigenicity”. This term refers the ability of transformed cells to form tumors in vivo. The in vitro experiments in this paper assess cellular transformation not tumorigenicity. These terms are distinct. The text should be changed accordingly. Another major concern is that the definition of oncogene addiction in this paper is vague. Soft agar assays are used to show that oncogene expression causes anchorage-independent growth. However, the effect of oncogene depletion is only tested under anchorage-dependent growth conditions. Will oncogene addiction be observed if KRAS and BRAF are depleted in cells grown in soft agar? This should be tested. Other concerns are noted below:

1. Figure 2B: why is the result inconsistent with published results? Please explain in more detail.

2. In all figures soft agar colony growth should be quantitated either as colony size or total colony number.,

3. Line 149-150: the reference to the A375 cell line as BRAF addicted is incorrect. Please change it.

4. Line 155: pERK does not change with siKRAS in H358 cells, which contradicts a previous report. It should be noted that H358 cells were grown in 10% FBS in this study, compared to 5% FBS for the Singh et al. study. Will the result change in lower serum conditions?

5. BRAF expression diminishes over time in MCF10A cells, which the authors state is an adaptation or negative feedback phenomenon. This is not a valid explanation. It could simply be a technical issue with the viral vector used to express BRAF, such as epigenetic silencing of the promoter. It is recommended to remove the data with late passage cells and include data from early passage cells to avoid confusion.

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Reviewer #1: No

Reviewer #2: No

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1 Mar 2021

We are grateful to all reviewers for the critical comments and useful suggestions that have helped improve our paper. We provide point-to-point answers to the comments and added detailed explanations in the revised manuscript. We put the comments made by the reviewer in bold italics with our responses below. Our changes in the manuscript are marked with yellow highlights.

Review Comments to the Author

Reviewer #1:

1. Assessment of oncogenicity:

For a tumour or cell line to be oncogenically addicted it must be shown to be oncogenic and that the removal of the oncogene then prevents oncogenicity. (a) the authors never show that these cells are capable of forming tumours in recipient animals. While soft agar assay positivity does correlate with tumor formation this is not always the case. (b) The bigger concern however, is that there is no use of soft agar assays (ie transformation assay) to determine whether the cells remain transformed after knockdown of the oncogene in question. This undoubtedly is because siRNA pools were used, which while being a standard first start to addressing a requirement, fail in any longer-term assessment of requirement. Longer term experiments can be accomplished through the use of lentiviral/retroviral vectors (even with inducible knockdown) that are completely compatible with longer term assays and make the cell population more uniform. Additionally, for BRAF the use of BRAFV600E specific drugs could have been employed in longer assays. Both approaches would work for tumorgenicity assays and soft agar transformation assays (if the authors do not have access to resources necessary for the mouse work). Without these, the only thing that the authors are able to study is a deviation in cell number and as proliferation in culture is not reflective of transformation or oncogenicity this makes statements about addiction difficult to assess. For example, while it is true that a severe reduction in proliferation would most likely cause a decrease in tumorigenicity, many proliferating cells are not themselves transformed - for instance the parental MCF10A cells are proliferative but not tumorigenic. Thus, if one uses proliferation as the sole assay for oncogene addiction then you are likely to mischaracterize phenotypes. Since the authors haven’t measured transformation as a readout then they really cannot say whether or not their cells are oncogene addicted. I encourage the authors to either assess this using lentiviral knockdown or alter the text considerably to reflect what they are assessing here (growth factor independence effects on proliferation).

We would like to appreciate the reviewer for his/her valuable comments. We agree with the critics pointed out by this reviewer, and therefore we have examined lentiviral-mediated knock-down of KRAS or BRAF and its effect on the soft agar colony formation assay during these three months.

Initially, we tried lentiviral-mediated knock-down, using the same target sequence of shRNA for KRAS gene as used by Singh et al (please see the Methods in this rebuttal for details).

We determined the titler of these lentiviruses (see Methods) and confirmed the effect of knock-down by western blotting (Figure 1 for reviewer only). The first problem was that shRNA-mediated knock-down of KRAS was not effective in MCF-10A/KRAS G12V OE cells (red arrows in Figure 1). This could be due to the high expression of KRAS in the cell line.

Figure 1 for reviewer only. Knock-down of KRAS or BRAF through lentivirus-mediated shRNA. (A) The indicated cells seeded on 12 well plates were infected with lentivirus expressing scramble shRNA (sh-Scr), shRNA for KRAS gene (sh-KRAS-1), or shRNA for BRAF gene (sh-BRAF-1). One day after infection, their culture medium was replaced with a fresh culture medium with 2.0 μg/mL puromycin to remove uninfected cells. Two to three days after puromycin selection, Cell lysates were prepared with 1xSDS sample buffers of varying volumes so that the number of cells was comparable, and subjected to western blotting. (B) The KRAS, BRAF, and pERK levels are plotted as bar graphs in each cell line. These expression levels of KRAS, BRAF, and pERK1/2 are divided by ERK1/2, followed by the normalization by dividing by the value of sh-Scr. Of note, shRNA for KRAS gene had no impact on the knock-down of KRAS proteins in MCF-10A/KRAS G12V OE cells.

The second problem was that all cell lines including MCF-10A/KRAS G12V OE cells gradually died after infection of lentivirus expressing shRNA for KRAS gene, even though this shRNA did not exhibit KRAS knock-down in MCF-10A/KRAS G12V OE cells (Figure 2 for reviewer only). This suggests the non-specific cytotoxicity of the lentivirus expressing shRNA for KRAS gene. We considered that the soft agar colony formation assay would not be suitable under these conditions.

Figure 2 for Reviewer only. Morphology of cells infected with the lentivirus expressing shRNAs. The cells were seeded in 6-well plate to a cell density of 9x10^4 cells/well (MCF-10A/KRAS G12V/+), 1.2x10^5 cells/well (MCF-10A/BRAF V600E/+), 1.0x10^5 cells/well (MCF-10A/KRAS G12V OE), and 1.2x10^5 cells/well (MCF-10A/BRAF V600E OE). One day after seeding, the cells were infected with the lentivirus expressing control shRNA (sh-Scr, scramble), shRNA for KRAS gene (sh-KRAS), or shRNA for BRAF gene (sh-BRAF) with polybrene. One day after infection, the medium was replaced with fresh medium + 2.0 μg/mL puromycin to select infected cells. Three days after selection, the cells were imaged with Olympus DP21 equipped with 4x objective lens.

For these reasons, we gave up long-term knock-down of KRAS or BRAF genes by lentivirus, and instead, we used siPOOLs to knock-down KRAS or BRAF, followed by soft agar colony formation assay. As this reviewer pointed out, the effect of siPOOLs-mediated knock-down does not last for a long time, and therefore we presume that siPOOLs-mediated knock-down affects the effect of cell survival and proliferation in the early stages of the soft agar colony formation.

In cancer-derived cell lines, we found that the number of colonies in A549 cells (non-addicted) and H358 cells (KRAS-addicted) was significantly reduced by KRAS knock-down (Fig. 3C and 3D). On the other hand, in A358 cells (BRAF-addicted), the number of colonies decreased by the knock-down of BRAF (Fig. 3C and 3D). These results seem to be consistent with the results of the proliferation assay.

Because three MCF-10A cell lines including parental, empty vector, and BRAF V600E OE late (data not shown) were incapable of soft agar colony formation in the first place (Figs. 2B-2E and 5B-5E), we did not perform soft agar colony formation assay with knock-down experiments on these cells. Thus, we investigated the effect of KRAS or BRAF knock-down on soft agar colony formation in the other three MCF-10A cell lines including KRAS G12V/+ cells, BRAF V600E/+ cells, and KRAS G12V OE cells. As a result, KRAS knock-down highly reduced the number of colonies in all three cell lines, and BRAF knock-down also decreased the number of colonies in all three cell lines, especially in BRAF V600E/+ cells (Figs. 4F, 4G, 6F, and 6G). From these results, we could evaluate the roles of KRAS and BRAF in the tumorigenic activity of these cells (Fig. 7). These results have been included in the revised manuscript and figures (Figs. 3C, 3D, 4F, 4G, 6F, and 6G).

On the other hand, we consider that these results are not suitable for the evaluation of oncogene addiction. This is because control MCF-10A cells (parental and empty vector) do not proliferate in the soft agar colony formation assay. If oncogene addiction has been acquired, we expect that knock-down of the oncogene would result in phenotypes such as apoptosis in oncogene addicted cells, but not in normal cells and oncogene non-addicted cells. However, as with cell proliferation in starvation medium (Figs. 4C-4E, and 6C-6E), the soft agar colony formation assay does not meet this criterion because control cells do not proliferate in the first place, and therefore these results were not used to evaluate oncogene addiction.

2. Choice of cells for this study:

This reviewer is slightly concerned about the use of MCF10a cells as a surrogate of normalcy. Despite being widely used, MCF10A cells do have properties that distinguish them from primary mammary epithelial cells (immortality as well as genomic rearrangements Cancer Genet Cytogenet, 2005 Nov;163(1):23-9.). But this concern is eclipse by the concern that the authors chose to study the effects of KRAS/BRAF mutations in breast epithelial cells. A quick search of the TCGA reveals that in ~6800 breast tumors 62 patients had 62 mutations in one or both of these genes (ie approximately 1% of breast cancer samples). One wonders why there is a lack of RAS/RAF mutations. Are mammary cells simply resistant to hyperactivation of the RAS/RAF? Are they sensitive to such signals and unduce senescence? Indeed, there is data to support both notions (Nature Cell Biol volume 9, pages 493–505 (2007)). The authors should state why they used this particular cell line for their studies in the text and in the discussion, what the caveats are for using such lines (vs normal mammary epithelial cells).

We would like to thank the reviewer for his/her effort to investigate the RAS/RAF mutation in mammary epithelial cells, and reasonable suggestions. According to the suggestion, we have included the following explanations in the revised manuscript:

Page 9, line 230

“This is because MCF-10A was spontaneously immortalized without defined factors [26], the cell line is not tumorigenic, i.e., they are not able to grow under anchorage-independent conditions or to form tumors when injected subcutaneously into nude mice [27], it does not have mutations in KRAS and BRAF, and it is easy to culture. In addition, it is beneficial to use MCF-10A because of the availability of cell lines with KRAS G12V and BRAF V600E as we mention below. On the other hand, it has been reported that MCF10A lacks a tumor suppressor gene, p16, which may render the cell line immortalized. Therefore, it should be noted that it is substantially different from normal mammary epithelial cells (see Discussion).”

Page 24, line 531

“The higher expression of KRAS G12V in mouse mammary epithelium in vivo has been found to induce cellular senescence in the p53-p16 pathway dependent manner [40]. However, homozygous loss of p16 in MCF-10A cells has been demonstrated [41], which probably makes the cells immortal and resistant to cellular senescence caused by KRAS G12V overexpression. These results strongly suggest a dose-dependent switch between cellular senescence and tumorigenesis of KRAS G12V in mammary epithelial cells. “

3. Data quality, presentation analysis:

a) The supplemental data supporting knockdown in the cancer derived lines and MCF10A-derivatives was rather weak. These are not very convincing blots (specifically those in Supp fig S2/S3). It is unclear how many times these were done. Quantification would be bolstered by repetition and statistics. In particular a direct comparison of MCF10A-derivatives should be presented side-by-side on one blot.

We agree with the reviewer’s comment that we require to demonstrate the repetition and statistics in western blotting data. We have included individual data points in all experiments including western blotting. The sample number, experimental condition, and statistical significance are described in the legends of those figures. In addition, we have included the data showing a direct comparison of expression levels of KRAS, BRAF, and ERK in all cell lines used in this study in Supplementary Figure S1C-E.

b) Are the blots within a given panel at the same levels of exposure? For example, figure 1D shows quantification relative to parental controls in full growth media but the blots are shown as being separate. In other situations (FgiS3a) the blots are exposed differently.

In all western blotting experiments, we detect the signal by near-infrared fluorescence with Odyssey CLx Infrared Imaging System (LI-COR), not conventional chemiluminescence. Therefore, the intensity of the excitation light and the scanning speed during detection affect the intensity of the signal. The conditions for this experiment are described in Materials and methods as follows:

Page 7, line 187

“The detection conditions are as follows: Resolution, 168 μm (Figs. 2F, 5F KRAS and BRAF, S3C, S4A, and S5C) or 84 μm (Figs. 5F empty vector, S1D, S2A, S2C, S3A, and S5A); sensitivity (scanning speed), normal (Figs. 2F, 5F KRAS and BRAF, S3C, S4A, and S5C) or low (Figs. 5F empty vector, S1D, S2A, S2C, S3A, and S5A).”

c) Figures involving protein quantification (2d, 4D) and cell number (3b,d,e,g5bce) should display that actual data points in addition to a graph with error bars) AND indicate statistical significance where present (with a statement or indicator where it is not). This would allow the reader to more accurately assess the data presented.

According to the reviewer’s suggestion, we have shown individual data points of all experiments and results of statistical tests in the revised figures.

This reviewer was surprised by the statement that the MCF10A-derived cells grew too fast to “reproducibly quantify the effect of RNAi on KRAS or BRAF ablation”. Do these cells grow faster than A549s or the other tumor derived cell lines? If not, the issue would most likely be due to transfection efficiency.

We apologize for the reviewer’s confusion, and please allow us to add an explanation. The cell proliferation rate of MCF-10A cells tends to be strongly dependent on cell density. Therefore, the seeding density at the time of cell passage has a great impact on the assay even a few days after the passage. It was quite difficult to determine the seeding cell number; if it is too small, all cells will die, and if it is too large, the cells will be overconfluent. In addition, the strong cell-to-cell adhesion of MCF-10A cells makes it difficult to quantitatively count the number of cells at the time of passage. For these reasons, we have tried to increase the reproducibility by reducing the cell proliferation rate with the partial growth medium, and have successfully obtained data. We have added these important notes in the revised manuscript as follows:

Page 14, line 340

“This is because the seeding density at the time of cell passage has a great impact on the assay even a few days after the passage.”

4. Finally this reviewer is concerned about inconsistencies with the published literature. MCF10A cells with KRAS G12D knockin mutations have different properties than the cells used here (the authors point this out). Are these cells different or is the same source? (the ones used in this paper are purchased) If they are the same cells, then why would there be a difference? If they are different cells, then what could be an explanation that could reconcile these data? The use of siRNAs to KRAS do not replicate data seen by others (including Jeff Settelman’s group). Again, this is perplexing and without explanation.

The MCF-10A/KRAS G12V/+ cells that we used were purchased from a commercially available source and are probably identical to the cells reported previously (Konishi, et al., 2007). Although the paper by Konishi et al. does not describe the details of the soft-agar colony formation assay experiment, it seems to be almost the same as our experimental conditions. The obvious differences are the number of seeding cells (2x10^4 cells vs. 3x10^4 cells), the interval between medium additions (every 3 days vs. every 1 week), and incubation time (4 weeks vs 3 weeks). We could not exclude the possibility that these differences might cause inconsistent results. Therefore, we have added these explanations in the revised manuscript as follows:

Page 10, line 249

“This result is not consistent with the previous report using the same cells [28], possibly because of the difference of the experimental conditions under which the seeding cell number (3x10^4 cells vs 2x10^4 cells), the top layer agar concentration (0.3% vs 0.4%), the interval of medium addition (every 3 days vs every 1 week), and/or incubation time (4 weeks vs 3 weeks).”

The difference between our results and those of Jeff Settleman and colleagues, i.e., the lack of decrease in pERK by knock-down of KRAS gene in H358 cells, could be explained simply by the difference in experimental systems. We cultured H358 cells in RPMI supplemented with 10% FBS, while Singh et al. had cultured the cells in RPMI supplemented with 5% FBS, as pointed out by Reviewer #2. Indeed, we confirmed the reproducibility that ERK phosphorylation was reduced by KRAS knock-down in 5% FBS culture medium (Fig. S2C and 2D). We have included this result in the revised manuscript.

Page 12, line 299

“In A375 cells, knock-down of the BRAF gene caused a concomitant decrease in both pERK and cell proliferation, which was consistent with previous reports showing that cancer cells harboring BRAF V600E are addicted to ERK pathway (S2A and S2B Fig)[32]. In H358 cells cultured in RPMI supplemented with 10% FBS, knock-down of the KRAS gene suppressed cell proliferation but not pERK with statistical significance (S2A and S2B Fig). Meanwhile, we could reproduce that ERK phosphorylation was significantly reduced in H358 cells cultured in 5% FBS medium as previously shown (S2C and S2D Fig)[16].”

5. Minor Text issues:

Line 134 reads, “…mutation of BRAF V600E in MCF-10A cells enhances in vitro tumorigenic activity… ”. The authors do not measure tumor formation, they are measuring transformation in culture. The text should be revised.

Figure 6 uses the word “tumorigenisities ”. I am not sure this is a word.

We have corrected these mistakes.

Reviewer #2:

This paper by Ito et el. uses cell line systems to investigate the induction of oncogene addiction following endogenous or exogenous expression of mutant KRAS(G12V) or BRAF(V600E). The authors conclude that expression of single mutant oncogenes in defined in vitro cell culture models does not lead to oncogene addiction.

We would like to thank the reviewers for the evaluations and critical comments.

Unfortunately, the data as presented do not support the major conclusion of the paper. Firstly, as the authors acknowledge (lines 250-251), KRAS or BRAF mutations are rarely found in breast cancer. Therefore, the use of MCF10A mammary epithelial cells as the only model for the experiments limits the ability to generalize conclusions. Additional cell line models should be tested using lung and pancreas-derived cell lines.

We agree with the reviewer’s comment, but it is unlikely that the same experiment could be performed in lung and pancreas-derived cell lines due to the time limitation. We would like to emphasize that this paper is a first step toward the understanding of molecular mechanisms of how cells acquire the property of oncogene addiction.

Secondly, the authors constantly refer to “tumorigenicity”. This term refers the ability of transformed cells to form tumors in vivo. The in vitro experiments in this paper assess cellular transformation not tumorigenicity. These terms are distinct. The text should be changed accordingly.

We agree with this comment, and according to the reviewer’s suggestion, we have replaced the term “tumorigenicity” with “transformation activity” throughout the revised manuscript.

Another major concern is that the definition of oncogene addiction in this paper is vague. Soft agar assays are used to show that oncogene expression causes anchorage-independent growth. However, the effect of oncogene depletion is only tested under anchorage-dependent growth conditions. Will oncogene addiction be observed if KRAS and BRAF are depleted in cells grown in soft agar? This should be tested.

We agree with the reviewer’s comment. This comment is exactly the same as Comment 1 raised by the reviewer 1. Please refer to the answer to Comment 1 from the reviewer 1.

1. Figure 2B: why is the result inconsistent with published results? Please explain in more detail.

We agree with the comment, and have included the explanation as follows:

Page 10, line 249

“This result is not consistent with the previous report using the same cells [28], possibly because of the difference of the experimental conditions under which the seeding cell number (3x10^4 cells vs 2x10^4 cells), the top layer agar concentration (0.3% vs 0.4%), the interval of medium addition (every 3 days vs every 1 week), and/or incubation time (4 weeks vs 3 weeks).”

2. In all figures soft agar colony growth should be quantitated either as colony size or total colony number.

According to the reviewer’s suggestion, we have quantified the data obtained by soft-agar colony formation assay as described in Materials and Methods. Briefly, we quantified the number of colonies that exceeded a certain threshold size and the average size of colonies. These data have been included in the revised manuscript and figures (Figs. 2E, 3D, 4G, 5E, and 6G).

3. Line 149-150: the reference to the A375 cell line as BRAF addicted is incorrect. Please change it.

We would like to thank the reviewer. We have corrected the reference.

4. Line 155: pERK does not change with siKRAS in H358 cells, which contradicts a previous report. It should be noted that H358 cells were grown in 10% FBS in this study, compared to 5% FBS for the Singh et al. study. Will the result change in lower serum conditions?

According to the reviewer’s suggestion, we examined the pERK level with siRNA for KRAS and BRAF in H358 cells cultured in 5% FBS as did in Singh et al. We could reproduce their results showing that siRNA for KRAS significantly reduced pERK level in H358 cells (Fig. S2C and S2D). We have included this result in the revised manuscript as follows:

Page 12, line 299

“In H358 cells cultured in RPMI supplemented with 10% FBS, knock-down of the KRAS gene suppressed cell proliferation but not pERK with statistical significance (S2A and S2B Fig). Meanwhile, we could reproduce that ERK phosphorylation was significantly reduced in H358 cells cultured in 5% FBS medium as previously shown (S2C and S2D Fig)[16].”

5. BRAF expression diminishes over time in MCF10A cells, which the authors state is an adaptation or negative feedback phenomenon. This is not a valid explanation. It could simply be a technical issue with the viral vector used to express BRAF, such as epigenetic silencing of the promoter. It is recommended to remove the data with late passage cells and include data from early passage cells to avoid confusion.

As the reviewers mentioned, we agree that we could not exclude the possibility that the adaptation of BRAF signaling is attributed to the gene silencing when oncogenic BRAF is expressed for a long time. However, we decided not to omit these data because we considered it would be more beneficial to the readers and the scientific community to include them in the paper honestly. Instead, we have added the following explanation in the revised manuscript regarding the possibility of gene silencing as pointed out by the reviewer:

Page 17, line 400

“During the course of experiments, we recognized that long-term culture of BRAF V600E OE cells reduced the expression levels of BRAF V600E and ERK phosphorylation levels (S4A Fig), probably due to the adaptation by a reduction of BRAF V600E expression through gene silencing and/or negative feedback mechanisms [33]”

Methods for Reviewers

Plasmids

pLKO.1-scr (scramble), pLKO.1-hKRAS, and pLKO-hBRAF. Plasmids were constructed in accordance with the standard molecular biology methods. The target sequences of the pLKO.1 vectors are as follows: scr, 5’-CCTAAGGTTAAGTCGCCCTCG-3’; hKRAS, 5’-CAGTTGAGACCTTCTAATTGG-3’ (Singh et al., 2009); and hBRAF, 5’-CCGGCAGCTTTCAGTCAGAT-3’. pLKO.1-TRC cloning vector was a gift from Dr. Root (Addgene plasmid # 10878).

Lentivirus-mediated shRNA expression

For delivery of shRNA via lentivirus, pLKO.1 vector was transfected into Lenti-X 293T cells (Clontech) together with the psPAX2, and pCMV-VSV-G-RSV-Rev by using the linear polyethyleneimine “Max” MW 40,000. Two days after transfection, the virus-containing supernatant was collected and filtered to remove 293T cells. The virus-containing supernatant was concentrated 20-fold by the Lenti-X concentrator (Clontech), and stored in a deep freezer. The cells were infected with the lentivirus in the presence of 8 μg/mL polybrene for 24 hrs, the virus-containing medium was replaced with a fresh culture medium with 2 μg/mL puromycin (InvivoGen), and the cells were further cultured for two or three days. The amount of virus to be infected was determined using HeLa cells, which showed no effect on KRAS or BRAF knock-down. Briefly, cells were seeded into 96 wells, and on the next day, the medium was replaced with the medium containing titrated lentivirus and 8 μg/mL polybrene. One day after infection, the virus-containing medium was changed to medium in the presence of 2 μg/mL puromycin. After two days, the number of survived cells was estimated by crystal violet staining. If the amount of virus was too high, the number of surviving cells decreased due to the virus toxicity, and if the amount of virus was too low, the number of infected cells decreased and most uninfected cells died by puromycin. Therefore, we determined the optimal amount of virus that showed the largest number of surviving cells.

Submitted filename: Response_to_Reviewers.docx

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18 Mar 2021

Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction.

PONE-D-20-30780R1

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22 Mar 2021

PONE-D-20-30780R1

Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction.

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