CaMKK2 Promotes the Progression of Ovarian Carcinoma through the PI3K/PDK1/Akt Axis.

Objective: To explore the functional role of Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in the progression of ovarian carcinoma (OC).
Methods: RT-qPCR analysis and western blot were conducted to detect the mRNA and protein expression of CaMKK2, PI3K, PDK1 and Akt in OC tissues and cells, respectively. CCK-8 assay, transwell migration assay and flow cytometry were used to measure cell proliferation, migration and apoptosis, respectively.
Results: CaMKK2, PI3K, PDK1 and Akt were highly expressed in OC tissues compared with the corresponding controls. CaMKK2 knockdown significantly suppressed the mRNA and protein expression of PI3K, PDK1 and Akt in HO8910 and OV90 cells. Moreover, CaMKK2 knockdown could dramatically repress cell proliferation, migration, and markedly elevate cell apoptosis in HO8910 and OV90 cells. Conclusions: CaMKK2 played a promotion role in OC progression via activating the PI3K/PDK1/Akt axis.

1. Introduction

Ovarian carcinoma (OC) is the deadliest gynecological malignancy derived from the ovary [1]. The incidence and mortality rates of OC are perpetually high with over 20,000 new cases diagnosed with OC and about 14,000 OC-related deaths annually only in USA [2]. Approximately 60 to 70% of OC patients were firstly diagnosed at the advanced stage due to lack of the typical symptoms [3]. It was reported that about 80% of OC patients developed resistance to metastasis and treatment [4, 5]. Despite the advancements have gained in chemotherapeutic, radio therapeutic and surgical treatment, the 5-year survival of OOC patients was still unsatisfactory due to the frequent recurrence, while the morbidity and mortality were elevated annually [6-8]. Therefore, identification of new diagnostic and prognostic biomarkers is a vital objective for OC treatment, which may be able to distinguish patients with OC at a high relapse risk and to explore biomarkers that are possible therapeutic targets for OC.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is an essential serine/threonine protein kinase that participates in many physiological processes [9]. CaMKK2 was proved to be upregulated in various cancers, such as hepatocellular carcinoma [10], prostate cancer [11] and gastric cancer [12]. More importantly, CaMKK2 was involved in human cancer biological behaviors, like cell multiplication and apoptosis. For instance, inhibition of CaMKK2 expression reduced tumor growth in prostate cancer xenotransplantation models [13]. In addition, the increase of CaMKK2 was also found in OC [14]. Thus, we explored the functional role and biological function of CaMKK2 in OC progression.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway is a main signaling network involving in normal and neoplastic cell growth and survival and plays an oncogenic role in multiple cancer types, including OC [15]. Phosphatidylinositol 3,4,5-trisphosphate synthesized by PI3K can recruit phosphoinositide-dependent kinase 1 (PDK1) and Akt to the plasma membrane, causing PDK1 phosphorylation of Akt. The activation of Akt resulted in enhancement of protein translation, cell survival, and cell growth [16]. The phosphorylated Akt regulated the expression of downstream apoptotic factors, thereby inhibiting cell apoptosis [17]. A previous study reported that the PI3K/PDK1/Akt axis is the key signal transduction pathway to inhibit apoptosis [18]. Moreover, the PI3K/PDK1/Akt pathway was proved to regulate epidermal growth factor-induced cell migration in SKOV3 and HO8910 cells [19]. However, the role of the PI3K/PDK1/Akt pathway in OC progression and whether CaMKK2 could regulate the PI3K/PDK1/Akt to participate in OC development need to be investigated.

In this study, we aimed to investigate the action of CaMKK2 and the PI3K/PDK1/Akt axis, as well as their potential mechanism in OC development.

2. Material and methods

2.1. Tissue samples acquirement

60 pairs of cancerous tissues and the adjacent non-cancerous tissues were acquired from 60 OC patients at Taizhou Hospital of Zhejiang Province affiliated to Wenzhou Medical University from February 2019 to February 2020. Participates provided the informed consents. All OC patients did not receive any treatment before participating in this research.

2.2. Cell culture and transfection

Two OC cell lines (HO8910 and OV90 cells) were obtained from Hunan Fenghui Biotechnology Co., Ltd. (Hunan, China). HO8910 and OV90 cells were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) containing 10% FBS in an incubator at37°C with 5% CO2. SiRNA targeting CaMKK2 (si-CaMKK2; 5'-GTGTTTACACAGTAAGATCAAGA-3') or CaMKK2-NC synthesized by Genepharma (Shanghai, China) were transfected into HO8910 and OV90 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

2.3. RT-qPCR analysis

TRIzol reagent (Yuanye Biotechnology Co., Ltd., Shanghai, China) was used to isolate the total RNA. Then, cDNA was synthesized using the First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Then, qPCR was performed using SYBR Green Master Mix kit (TaKaRa) on the ABI7500 fluorescence quantitative PCR instrument (Long Jump Biological & Science Technology Development, Beijing, China). Finally, the mRNA levels of CaMKK2, PI3K, PDK1 and Akt were calculated using the 2-∆∆Ct method, with β-actin as the internal reference gene. The primer sequences were synthesized by Sangon Biotechnology Co., Ltd., (Shanghai, China) and listed in Table 1.

Table 1

The primer sequences of the related genes.

	Upstream	Downstream
CaMKK2	5′-TAAAGACCATGATTCGAAAG-3′	5′-CTTTCACAAGAGCACTTC-3′
PI3K	5′-TTCCCTCGCAATAGGTTCTCC-3′	5′-GACCAATACTTGATGTGGCTGAC-3′
PDK1	5′-TGAACTGACCTTGCCACAT-3′	5′-TGAAGCAGCACTGAACACG-3′
Akt	5′-CATGAGGATCAGCTCGAACAGC-3′	5′-ACGGGCACATCAAGATAACGG-3′
β-actin	5′-CCGTTCCGAAAGTTGCCTTTT-3′	5′-ATCATCCATGGTGAGCTGGC-3′

2.4. Western blot

Total protein was extracted from OC tissues and cells using RIPA buffer (Beyotime, Shanghai, China). The protein concentration was determined by BCA kit (TaKaRa). After 10% SDS-PAGE electrophoresis separation, the proteins were transferred on the PVDF membranes. Then, the membranes were blocked with 5% nonfat-dried milk for 1 h and incubated with the specific primary antibodies including CaMKK2 (1 : 1000, ab96531, Abcam, Cambridge, UK), PI3K (1 : 1000, ab32089, Abcam), PDK1 (1 : 1000, ab110025, Abcam), Akt (1 : 1000, ab8805, Abcam) and β-Actin (1 : 1000, ab6276, Abcam) at 4°C overnight. Subsequently, the membranes were washed and then incubated with the horseradish peroxidase-labeled goat anti-rabbit (1 : 2000; ab6721, Abcam) or goat anti-mouse secondary antibodies (1 : 5000, ab6789, Abcam) for 1 h at 37°C. Finally, the protein bands were presented using ECL (Millipore, Bradford, MA, USA) in the dark and the gray values were analyzed using Quantity One software.

2.5. Detection of cell multiplication

HO8910 cells and OV90 cells were seeded in the 96-well plates. After transfection, 20 μL of CCK-8 solution (Beyotime) was added to each well and incubated for 2 h. Then, cell proliferation was observed via detecting the OD value at 450 nm using the microplate reader (Image Trading Co., Ltd., Beijing, China).

2.6. Detection of cell migration

Transwell assay was used to evaluate cell migration. HO8910 and OV90 cells were added into the upper chamber (SunBio Biomedical Technology, Shanghai, China) with 200 μL RPMI-1640 medium without serum. The lower chamber (SunBio Biomedical Technology) was supplemented with 600 μL RPMI-1640 containing 20% FBS. 24 h later, the migrated cells on the bottom chamber was counted.

2.7. Flow cytometry

After transfection, HO8910 and OV90 cells were washed, digested and re-suspended in 400 μL 1 × binding buffer (Invitrogen). Then, the treated HO8910 and OV90 cells were stained with 10 μL AnnexinV/PI (Beyotime) without light. Finally, the apoptotic cells were detected by the CoulterCytoFLEX flow cytometry (Beckman Coulter, Miami, FL, USA). The experiment was repeatedly determined for 3 times.

2.8. Statistical analysis

SPSS 20.0 was used to process the collected experimental data. Measurement data was expressed as the mean ± standard deviation (SD). Student's t test was used for comparing the difference between two groups, and one-way ANOVA was utilized for comparison of data among multiple groups. Visualization of the experimental data was performed by Graph Pad Prism 7. Difference with P-values <0.05 was considered as statistically significant.

3. Results

3.1. CaMKK2 was increased in OC tissues

As shown in Figures 1(a)–1(d), the mRNA expression of CaMKK2, PI3K, PDK1 and Akt was significantly upregulated in tumor tissues in comparison to the adjacent normal tissues. Similarly, the protein expression of CaMKK2, PI3K, PDK1 and Akt was notably elevated in cancerous tissues (Figures 1(a)–1(e)).

Figure 1

CaMKK2 was increased in OC tissues. (A-D) The mRNA and protein expression of CaMKK2, PI3K, PDK1 and Akt were determined using RT-qPCR and western blot, respectively. (E) The representative images from western blot results. ∗P <0.05.

CaMKK2 knockdown inhibited the expression of PI3K, PDK1 and Akt in HO8910 cells.

The results from RT-qPCR analysis in Figures 2(a) and 3(a) showed the successful knockdown efficiency of si-CaMKK2 in HO8910 cells. The data demonstrated that downregulation of CaMKK2 decreased the mRNA and protein expression of PI3K, PDK1 and Akt in HO8910 cells (Figures 2(b)–2(d) and 3(b)–3(d), 3(f)). However, the protein expression of CaMKK2, PI3K, PDK1 and Akt was not changed in Figure 3(e).

Figure 2

CaMKK2 knockdown inhibited the mRNA expression of PI3K, PDK1 and Akt in HO8910 cells. (A-D) The mRNA expression of CaMKK2 (A), PI3K (B), PDK1 (C) and Akt (D) in HO8910 cells was assessed using RT-qPCR analysis. ∗P <0.05.

Figure 3

CaMKK2 knockdown inhibited the protein expression of PI3K, PDK1 and Akt in HO8910 cells. (A-D) The protein expression of CaMKK2, PI3K, PDK1 and Akt in HO8910 cells was determined using western blot. (E) The representative images from western blot results in HO8910 cells before transfection. (F) The representative images from western blot results in HO8910 cells after transfection. ∗P <0.05.

CaMKK2 downregulation suppressed PI3K, PDK1 and Akt levels in OV90 cells.

The successful efficiency of si-CaMKK2 was also verified in OV90 cells (Figures 4(a) and 5(a)). In addition, we also proved that knockdown CaMKK2 could downregulate the mRNA and protein expression of PI3K, PDK1 and Akt levels in OV90 cells (Figures 4(b)–4(d) and 5(b)–(d), 5(f)), while the protein expression of CaMKK2, PI3K, PDK1 and Akt was not affected in OV90 cells before transfection (Figure 5(e)).

Figure 4

CaMKK2 downregulation suppressed PI3K, PDK1 and Akt mRNA levels in OV90 cells. (A-D) RT-qPCR analysis was used to detect the mRNA expression of CaMKK2 (A), PI3K (B), PDK1 (C) and Akt (D) in OV90 cells. ∗P <0.05.

Figure 5

CaMKK2 downregulation suppressed PI3K, PDK1 and Akt protein levels in OV90 cells. (A-D) Western blot was performed to determine the protein expression of CaMKK2, PI3K, PDK1 and Akt in OV90 cells. (E) The representative images from western blot results in OV90 cells before transfection. (F) The representative images from western blot results in OV90 cells after transfection. ∗P <0.05.

CaMKK2 knockdown repressed cell multiplication and migration in HO8910 and OV90 cells.

Cell multiplication of HO8910 and OV90 cells were not evidently different before transfection (Figures 6(a) and 6(d)). CCK-8 assay revealed that cell multiplication was significantly inhibited by transfection of si-CaMKK2 in HO8910 and OV90 cells (Figure 6(b) and 6(e)). Moreover, we found that cell migration was dramatically attenuated by CaMKK2 knockdown in HO8910 and OV90 cells (Figures 6(c) and 6(f)).

Figure 6

CaMKK2 knockdown repressed cell multiplication and migration in HO8910 and OV90 cells. (A and C) Proliferation of HO8910and OV90 cells before transfection was detected by CCK-8 assay. (B and E) CCK-8 assay was utilized to assess cell proliferation of HO8910 and OV90 cells after transfection. (C and F) Cell migration was evaluated using transwell migration assay. ∗P <0.05.

CaMKK2 knockdown enhanced the apoptosis of HO8910 and OV90 cells.

As described in Figure 7, the apoptosis rate was similar in the two groups of HO8910 and OV90 cells before transfection, while transfection of si-CaMKK2 upregulated the apoptotic rate in HO8910 and OV90 cells.

Figure 7

CaMKK2 knockdown enhanced the apoptosis of HO8910 and OV90 cells. (A-B) Cell apoptosis in HO8910 and OV90 cells was detected using flow cytometry. (C and E) The representative images of flow cytometry in HO8910 and OV90 cells before transfection. (D and F) The representative images of flow cytometry in HO8910 and OV90 cells after transfection. ∗P <0.05.

4. Discussion

As the fifth leading cause of cancer-related death in female, OC was reported as one of the most lethal gynecological malignancies in the developed countries [1, 2]. Previous studies revealed that OC is easy to migrate and metastasize to the abdominal organs [20, 21]. Unfortunately, a small percentage of women were diagnosed with OC before it spread outside the ovaries [22]. Usually, the combination of taxanes and platinum-based chemotherapy was used for OC therapy. However, cancer cells developed drug resistance and remained dormant at the site of metastasis, leading to the recurrence in cancer [23]. Although the methods of early diagnosis and comprehensive treatment have achieved improvements, the recurrence and death rates remain disappointing in OC patients. The mechanism of OC progression was extremely complex, thus it might be of significance to find out a potential biomarker for OC patients diagnosis and treatment. Therefore, we investigated the action of CaMKK2 and its potential underlying mechanism in OC progression and aimed to find a possible target for OC therapy.

CaMKK2 acted as a vital regulator in types of cancers. In hepatocellular carcinoma and prostate cancer, CaMKK2 served as an attractive drug target downstream of AR, downregulation of CaMKK2 could eliminate tumor growth and inhibit macrophage-mediated inflammation [24]. In glioma, CaMKK2 was found to be highly expressed and facilitate cancer cell migration and multiplication, leading to disease deterioration [25]. In this study, we also found the elevated CaMKK2 in OC tissues. Moreover, CaMKK2 knockdown significantly inhibited cell multiplication, and migration, and facilitated cell apoptosis in OC cells. Additionally, the apoptosis rate of OC cells was dramatically facilitated after knocking down CaMKK2. A previous study on breast cancer revealed that CaMKK2 knockdown increased the rate of apoptosis in breast cancer cells [26], which was similar to the results of this study. Based on the abovementioned results, we disclosed that CaMKK2 exerted the oncogenic effects on OC progression.

In this study, we found that CaMKK2, as well as PI3K, PDK1 and Akt were increased in tumor tissues. In addition, the levels of PI3K, PDK1 and Akt were decreased after CaMKK2 knockdown in OC cells. Thus, we speculated that CaMKK2 was involved in OC development through regulating the PI3K/PDK1/Akt axis. Akt is one of the most common hyperactivated kinases in cancer, which played a pivotal role in promoting cell migration. Previous studies have found that inhibiting PI3K/Akt axis could lead to apoptosis of cervical cancer cells [27]. PI3K, a member of the heterodimeric lipid kinase family, is activated in conjunction with its related domain proteins AKT and PDK1 [28]. PDK1, a phosphoinositol, was significantly increased that could effectively activate Akt [29]. In a variety of malignancies, overexpression of the PI3K/PDK1/Akt axis could increase tumor mobility [30]. Generally, the PI3K/PDK1/Akt axis was reported to be used as a target of cancer [31]. Combined with the results of this study, PI3K/PDK1/Akt was decreased by knockdown of CaMKK2 expression, indicating that CaMKK2 exhibited the induction effect on the PI3K/PDK1/Akt axis in OC. A previous research revealed that the higher the level of CaMKK2 in clinical specimens indicated the more advanced the tumor [16]. CaMKK2 is an activator of Akt, and the development of its inhibitors contributed to the improvements of clinical therapies targeting the carcinogenic PI3K/PDK1/Akt axis [16]. Our results concluded that increase of CaMKK2 could activate the PI3K/PDK1/Akt axis in OC, thereby further worsening this cancer.

This study also had shortcomings. The number of tissues samples from OOC patients was a little small. Moreover, we will study the specific molecular mechanisms of OC in depth, such as the role of miRNAs in OC.

To sum up, CaMKK2 activated the PI3K/PDK1/Akt axis in OC, and the increased level of CaMKK2 lead to further deterioration of OC, providing a potential target for therapeutic approaches of OC.