Drug-Resistant Breast Cancer: Dwelling the Hippo Pathway to Manage the Treatment.

Breast cancer can be categorized as a commonly occurring cancer among women with a high mortality rate. Due to the repetitive treatment cycles, it has been noted that the patients develop resistance towards the chemotherapeutic drugs and remain unresponsive towards them. Therefore, many researchers are studying various signaling pathways involved in drug resistance for cancer treatment to overcome the obstacle. Hippo signaling is a widely studied pathway involved in tumor progression and controlling cell proliferation. Hence, understanding the aspects of the gene involved Hippo pathway would provide an insight into the mechanism behind the resistance and result in the development of new treatments. Here, we review the Hippo signaling pathway in humans and how the expression of different components leads to the regulation of resistance against some of the common chemo-drugs used in breast cancer treatment. The article will also discuss the chemotherapeutics that became ineffective due to the resistance and the mechanism following the process.

Introduction

Breast cancer can be described as a fatal disease as it contributes to increasing death rates in women worldwide and is also considered the second most common type of cancer next to lung cancer. If untreated or late-diagnosed, breast cancer can easily infilter to adjoining lymph nodes and metastasize to distant significant organs such as the liver, lung, and brain.1 According to the WHO statistics, in 2020, around 2.3 million women were diagnosed with breast cancer, besides 6,85,000 deaths globally.2 Cancer is life-threatening and remains incurable mainly because the symptoms are asymptomatic, leading to the early onset but the late prognosis. By the time cancerous cells were identified, the disease had already progressed in the patient’s body. Lagging prognosis leads to the development of local and distant metastasis depending on the nature of the tumorous growth.

Chemotherapy is one of the foremost and influential ways to treat breast cancer in most countries. However, it is essential to emphasize that while applying chemotherapy to the patient, the main hurdle that prevents the diminishing of cancerous growth is drug resistance. In this case, after a few treatment cycles, the patient starts to develop resistance against the supplied drug, thus limiting the patient’s benefits.3

Through the various studies conducted to modulate the process of drug resistance in cancer therapy, the Hippo pathway came to light. Hippo signaling proves to be an essential aspect in imparting drug resistance, leading to ineffectiveness in treatment. The hippo pathway is a signaling mechanism commonly witnessed in mammals and is involved in cellular processes at the genomic level leading to alteration in gene expression. Thus, the potential of genes in Hippo Signaling can be explored in significant mechanisms leading to malignancy and drug resistance in cancer therapy. Several recent data on various solid tumors have pointed out the alteration in the Hippo pathway, suggesting a possible association in pathogenesis and drug resistance.3–6 For example, in breast cancer, the co-transcriptional factors YAP and TAZ of the hippo pathway are deregulated with increased TAZ and YAP1. The raised expression of YAP1 and TAZ has been linked to Her2+, Luminal A, and Luminal B and triple-negative breast cancer (TNBC).7 Furthermore, the expression of YAP1 was also noticed to be linked with a low survival rate in HER2+ positive breast cancer patients. These potential pieces of evidence suggest significant crosstalk between the hippo pathway and the molecular biology of breast cancer.8,9 So, it is crucial to understand the fundamental mechanism to develop effective chemotherapeutic treatments.10

Hippo Signaling Pathway

The hippo signaling pathway includes the cascade mechanism responsible for controlling the size of the organs by maintaining cell growth and apoptosis in animals and humans.11 This pathway was first identified as a significant governor to regulate the organ’s size and was first discovered in Drosophila melanogaster.12 This led to the discovery of the 4 tumor suppressors which are; Warts (Wts),12,13 Salvador (Sav),3,14 Hippo (Hpo)15–19 and Mob-as-tumor-suppressor (Mats).20 These tumor suppressors work by forming the core kinase cascade, which controls cell proliferation and apoptosis.

Initially, activated Hpo phosphorylates and activates the Wts-Mats complex.17,21 At the same time, Sav, which is also activated when phosphorylated by Hpo, activates the Wts-Mats complex by acting as a protein scaffold.17,18

The downstream target of the activated Wts is Yorkie (Yki) which plays the role of a transcriptional co-activator.22 In the case of Yki, the DNA binding domains are mainly absent. Despite the absence of the DNA binding domains, Yki binds to the Scalloped (Sd), a DNA binding partner, to regulate the transcription of genes.23 Wts is capable of phosphorylating Yki in multiple sites, which can also be viewed as an effective regulatory mechanism to downregulate the expression of Yki. Phosphorylation at multiple sites leads to restricting the nuclear localization and cytoplasmic sequestration of Yki that transcriptionally inactivates the target gene.24–26

Meanwhile, in the absence of the signaling, Yki moves towards the nucleus and interacts with Sd, another transcriptional factor. This results in activating the expression of diverse genes, including cyclin E and diap1 (Figure 1A). The whole process results in increased expression, leading to cell proliferation and a decrease in cell apoptosis expression.19

Figure 1

Hippo pathway gene regulation in (A) Drosophila melanogaster (B) mammals.

In the case of human beings, the core elements that are involved in the mammalian Hippo pathway includes Serine/threonine kinases: Examples includes Mammalian sterile 20 like kinase1/2 (MST1/2), Large tumor suppressor1/2 (LATS1/2), Salvador Homolog 1 (SAV1), and MOB kinase activator 1A/B (MOB 1A/B).

It is important to note that MOB 1A/B acts as an adapter protein for MST1/2 and LATS1/2.2

In humans, the LATS1/2-MOB1A/B complex is phosphorylated and activated by MST1/2 – SAV1 complex, which results in the phosphorylation and inactivation of the Yes-associated protein (YAP) and co-activator TAZ, which are classified as the orthologs of the Yki in mammals.27 LATS1/2 works by phosphorylating YAP/TAZ at multiple sites and promotes its binding to 14-3-3 protein, leading to cytoplasmic retention. Phosphorylation also results in ubiquitination that directs the process of proteasomal degradation.27–29

The potential of hippo signaling is that the pathway is majorly responsible for controlling cell proliferation, apoptosis, and differentiation.30 If this pathway is altered due to the inactivation of tumor suppressor genes, the YAP-TAZ will move towards the nucleus, and once they reach the nucleus, they bind to the transcriptional factors of the TEAD family (Figure 1B) and activate the expression of genes that are responsible for promoting the cell proliferation and results in disrupted tumor growth and carcinogenesis.31

In conclusion, due to the genetic changes (gene silencing, transcriptional regulations, and mutations), the activity of the tumor suppressors is subdued, which then proceed to hyper activate the YAP/TAZ and develop different types of cancer.

Cancer Progression Due to Hippo Pathway

Hippo Pathway is primarily responsible for mediating cell proliferation, apoptosis, differentiation, and migration (Figure 2).32 In cancer, due to the inactivation of tumor suppressors such as LATS1/2, the pathway is not activated, it results in the hyperactivation of the YAP/TAZ, resulting in the increased expression of CRY61 and BCL2 family, which ultimately suppresses the apoptosis process as they are the factors responsible for promoting cell survival.26,33–35

Figure 2

Cellular roles of Hippo pathway.

In breast cancer, the upregulation of the YAP and TAZ (transcriptional co-activators) are mainly responsible for cancer initiation, growth, and metastasis. TAZ plays a significant part in cancer stem cells’ tumor initiation and self-renewal capacity.32 TAZ, in a way, enriches cancer originating stem cells which result in the formation of tumors.36 The luminal cells even start showing the characteristics of the basal cells due to the overexpression and activity of the TAZ oncoprotein, therefore resulting in the progression of basal-like breast cancer.32,36 YAP/TAZ mediates the activity of the other oncogenic components such as LIF and GPER. Increased expression of YAP results in the downregulation of the Leukemia Inhibitory Factor Receptor, which ultimately results in increased metastatic and invasive potential of the cells.37 The hyperactivation of YAP/TAZ results in the upregulation of the G-protein coupled estrogen receptor, which stimulates the tumor growth and movement of the cancerous cells to the surrounding tissues.32 When the Hippo pathway is not activated, the YAP/TAZ complex moves towards the nucleus. It binds to the TEAD transcriptional factors, resulting in the increased expression of many oncogenic factors such as CYR61 and CTGF, leading to progression in breast cancer.34,35,38 The hyperactivation of YAP results in elevated levels of KLF5 proteins, thus promoting cell proliferation and cell survival (Figure 3).39

Figure 3

Hippo pathway genes in breast cancer progression.

Treatment Strategies Used in Breast Cancer

Breast cancer belongs to the class of heterogeneous disease(s) and is basically divided into three subtypes depending on how the hormone receptors are expressed, which include estrogen and progesterone, Human epidermal growth factor receptor 2 (HER2), Triple-negative breast cancer.40

In breast cancer, the medicinal therapies involved are mainly chemotherapy, hormonal therapy, targeted therapy, and immunotherapy.

Chemotherapy destroys the cancer cells and shrinks the tumor growth before or after surgery. Drugs mainly used in chemotherapy for breast cancer treatment are listed (Table 1). Further, these drugs can be used in combination depending upon the medical oncologist.

Table 1

Commonly Used Chemotherapeutic Drugs for Breast Cancer Treatment

List of Drugs	Mode of Action*
Paclitaxel	A plant alkaloid with anti-microtubule activity.
Lapatinib	Synthetic drug that ceases EGFR phosphorylation.
Doxorubicin, Epirubicin	An anthracycline that blocks topoisomerase enzyme.
Cisplatin	Impede DNA replication.
Tamoxifen	Compete with E2 and block its targeted role.
5-Flouro Uracil	Restricts thymidine formation for DNA synthesis.
Capecitabine	Converts to 5-FU.
Carboplatin	Modifies the DNA molecule by forming reactive platinum complexes that inhibit DNA synthesis.
Cyclophosphamide	Crosslinking of RNA and DNA strands leading to protein synthesis inhibition.
Eribulin	Inhibit microtubules that are in their growth phases.
Gemcitabine	Inhibit DNA synthesis by blocking chain elongation.
Ixabepilone	Suppressor of microtubules.
Methotrexate, Amethoterin	Suppress DHFR enzyme activity that leads to DNA, RNA, and protein formation inhibition.
Protein-bound paclitaxel	Nanoparticle bound paclitaxel with the same activity as paclitaxel.

Note: *Curated from ACCC Oncology Drug Database.

It is important to note that each tumor grows in a specified environment to help its growth and transmission. The targeted therapy works by targeting the specific components promoting the growth and survival of the cancerous cells, such as the specific gene, protein, or hormone. The identified targets are then treated, which results in the blockage of the growth of tumor cells as their niche is destroyed.

For example, the targeted therapies used to treat HER2-positive breast cancer are Trastuzumab, Pertuzumab, Neratinib, and Ado-trastuzumab emtansine. In addition, the therapies mentioned above are used in conjugation to chemotherapeutics, including taxanes and anthracyclines, to treat triple-negative breast cancer.41

Drug Resistance Mediated by Hippo Signaling

Although cancer largely remains incurable due to various aspects, it is essential to emphasize that chemotherapy is one of the most effective treatments for cancer patients. However, the hurdle that occurs with antibiotics is that drug resistance is also seen in cancer therapy due to genetic and epigenetic mutations and the metabolic mechanism involved in drug inactivation and efflux hampering the patients from the treatment.3 Various studies emphasized that Hippo signaling can be vital in imparting drug resistance and ineffective treatment. Bortezomib, an inhibitor that interacts with the hippo pathway, is being analyzed in the clinical trial Phase III to treat different cancers, including breast cancer.42 In an earlier study, it had been noticed that breast cancer stem-like cells (BCSCs), a specific group of self-renewal cells, are thought to be accountable for drug resistance and cancer spread. Interestingly, when activated, the TAZ and YAP assist in maintaining breast cancer cells with stem cell-like properties.8

Drug resistance is mainly observed due to the upregulation or the downregulation of the components that play an integral part in the hippo signaling pathway (Table 2).

Table 2

Hippo Signaling Components and Their Associated Role in Drug-Resistant Breast Cancer

Hippo Signaling Component	Dysregulation	Drugs	References
YAP	Hyperactivation	Lapatinib, CDK 4/6 inhibitors, Paclitaxel, Doxorubicin	[43–54]
TAZ	Hyperactivation	Paclitaxel, Doxorubicin, Lapatinib, CDK 4/6 inhibitors	[43–45,47,48]
hEx	Downregulation	Paclitaxel, Taxol	[55]
MST	Downregulation	Cisplatin	[56]
LATS1	Downregulation	Taxol	[57,58]
LATS2	Downregulation	Doxorubicin, Tamoxifen	[59,60]

Paclitaxel

Paclitaxel is the drug administered to treat different types of cancer and is classified as the widely used drug in this domain. The drug is plant-derived and is extracted from the Taxus brecifolia. Paclitaxel works by binding to the β-tubulin, thus maintaining the spindle formation at the time of mitosis.61,62 Resistance to paclitaxel can ascribe to the hyperactivation of YAP/TAZ or the downregulation of hEx. YAP can be described as a transcriptional co-activator capable of functioning as an oncoprotein by interacting and activating other transcriptional factors.35,63 For example, in the cancerous growth hyperactivation of YAP-S127A due to the abbreviated phosphorylation site, the YAP reaches the nucleus, accumulates there, and produces resistance to paclitaxel.52

TAZ can be understood as an analog to YAP. Both YAP and TAZ are termed oncogenes, resulting in cell proliferation and cancerous growth.64,65 Overexpression of TAZ results in the development of resistance against paclitaxel as TAZ’s overexpression results in the increased levels of Multidrug resistance proteins (MDR). When activated, these proteins, the cell membrane transporters, reduce the drug concentration, resulting in anti-drug resistance.3

hEx is also responsible for controlling cell proliferation as it binds to the Yki.66 So, the downregulation of hEx results in the increased activity of Yki, resulting in the development of paclitaxel resistance (Figure 4).55 Further, Cyr61 and CTGF, the target for TAZ/TEAD, provide resistance to paclitaxel-mediated breast cancer treatment. Therefore, the activity of paclitaxel was reversed to its original form by inhibiting Cyr61 and CTGF with the short hairpin RNA method.45

Figure 4

Hippo signaling in regulating chemo-drugs resistance.

Lapatinib

Lapatinib is a therapeutic drug and works as a reversible inhibitor for tyrosine kinase regions of HER2. Epidermal growth factor receptor holds significance in treating advanced or metastatic breast cancer.

It works by competing with ATP to reversibly inhibit the ATP-binding pocket by forming weak interactions. As it binds to the pocket instead of ATP, this results in the downregulation and blocking of the targeted enzymes such as Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-kinase, Akt, and also results in inhibition of the mammalian target of rapamycin dependent transduction pathways, and this blocking results in the arresting the cell growth and finally results in the apoptosis of tumorous cells.67,68

It is a HER2 targeted kinase inhibitor, and it is observed that in vitro, after subsequent administration, the HER2 breast cancer cells develop resistance against the lapatinib.3 Furthermore, the association of this resistance development and hyperactivation of YAP and TAZ has been observed in previous studies. Therefore, it is believed that the outcome of decreased/minimal expression of YAP/TAZ can be positively correlated with increased sensitivity of lapatinib (Figure 4).31

CDK 4/6 Inhibitors

Cyclin-dependent kinase, also known as CDKs, helps in regulating the cell cycle. CDK 4 and 6 act as a network, manage the cell cycle, and controls cell proliferation. CDK 4 and CDK 6 works as a complex with D-type cyclins, and in the case of mitogenic stimulation, the response is produced as they work together to drive cell progressions from the G phase to the S phase.69,70

CDK 4/6 inhibitors are administered to treat oncogenic growth and are administered along with hormonal therapy, including the aromatase inhibitor or fulvestrant. These inhibitors are vital in treating hormone receptors, HER2, and advanced or metastatic breast cancer.3

However, their effectiveness is restricted due to the drug resistance imparted by the hyperactivation of YAP and TAZ.31 In ER-positive cases, the loss of FAT1 steered the hyperactivation of YAP/TAZ as the Hippo pathway is suppressed. As the suppression of pathway directs the increased expression of the YAP/TAZ, they cluster around the CDK6 promoter and facilitate the CDK 6 transcription. Additionally, increased expression of CDK6 imparts resistance to the CDK 4/6 inhibitors in the case of the breast cancer cells and the inactivation of FAT1 protein (Figure 4).3

Tamoxifen

It is an estrogen receptor modulator, and it is utilized to medicate breast cancer.44 People diagnosed with breast cancer express the ERα protein and respond to ERα antagonists.75,76

However, the resistance to hormone therapy is observed in patients and tamoxifen resistance is developed due to the downregulation of LATS2.69 The downregulation of LATS2 results in its co-localization with the ERα in the nucleus. As LATS2 can activate the ERα transcription, it promotes the increased expression of YAP, which in turn increases the transcription of estrogen receptor alpha, causing inhibition of the action of tamoxifen drug.57,60 Consequently, regulating the expression of LATS2 protein can overcome this resistance towards tamoxifen.

Doxorubicin

Doxorubicin falls under the category of anthracycline drugs and works by binding to the enzyme topoisomerase II and blocking its action. Since this is the enzyme that the cancerous cells require to grow and divide, the binding of doxorubicin to the topoisomerase enzyme results in cell apoptosis. Therefore, this drug is commonly administered to treat various kinds of cancer.71

Doxorubicin resistance is developed due to the hyperactivation of YAP/TAZ and the decreased expression of the LATS2. In the case of YAP, its overexpression leads to the partial actuation of the MAPK (Mitogen-Activated Protein Kinase) pathway, which promotes cell proliferation.3 It is also noted that YAP and p53 are activated when the cells accompanied by doxorubicin are subjected to treatment. Hence the activation and overexpression of wildtype YAP promote the development of resistance.72 Also, p53, in a way, controls the expression of the YAP where YAP binds to the promoter of p53 to maintain the apoptosis, whereas p53 by binding to YAP’s promoter increases its expression, thus regulating its function.72

In the case of TAZ, its hyperactivation leads to the activation of the interleukin-8 (IL-8), and the increased levels of MDR proteins in Ras-transformed MCF10A-T1K cells leads to the development of resistance.3,35

LATS2 is responsible for maintaining the expression levels of YAP/TAZ in the Hippo Pathway by phosphorylating the YAP/TAZ component to lead to cytoplasmic retention, ubiquitination, and eventually proteasomal degradation. In the case of breast cancer, the LATS2 levels are downregulated, and due to this, it is unable to phosphorylate the YAP component, which results in hyperactivation due to overexpression of the YAP component and further leads to drug resistance in cells.54

Cisplatin

Cisplatin falls under the category of chemotherapeutic drug and holds the utility to treat diverse classes of cancer. It works by forming bonds with DNA, causing damage which results in apoptosis.45 Therefore, cisplatin drug is always administered with paclitaxel as the first-line treatment.

It is found that in cisplatin resistance breast cancer cells, downregulation of the MST protein is observed. MST is an integral part of the Hippo signaling pathway, and MST 1/2 is responsible for phosphorylating YAP and TAZ and downregulating them. Through autophosphorylation, MST1/2 gets activated, which in turn phosphorylates downstream LATS kinase that helps make LATS further phosphorylate the YAP and TAZ, consequently controlling cell proliferation.73,74 Overall, the hyperactivation results in the impartment of the cisplatin resistance on the patients.55

Taxol

Taxol is a therapeutic drug and is commonly used to treat breast cancer patients. The primary role of taxol is the induction of apoptosis to destroy cancerous growth.

Again, the TAZ protein is found to be overexpressed in breast cancer cells showing resistance towards chemo-drug Taxol. Highly expressed TAZ molecules move towards the nucleus and interact with the TEAD proteins. TEAD factors are responsible for promoting the various TAZ functions.32,33

Since TAZ is a transcriptional co-activator, its increased levels mark the activation of various downstream targets, resulting in the activation and increased expression of many oncogenic factors such as CYR61 and CTGF. The TEAD response elements bind to the promoters of CYR61 and CTGF and support their elevated expression. This ultimately suppresses the apoptosis process as they are the factors that are responsible for promoting cell survival and proliferation and interrupt the cancer treatment (Figure 4).27,35–37,45

Conclusion

The development of drug resistance proves to be a significant setback for chemotherapy as it is the most commonly used procedure to treat cancer patients. The molecules involved in the hippo signaling pathway play a vital role in developing this resistance. The deregulation of the hippo pathway, be it the inactivation of tumor suppressor genes LATS1/2 or the increased expression of oncogenes YAP/TAZ, results in the disrupted expressions of downstream targets causing the cancer cells to develop resistance against the anti-cancer drugs. A recent study identified the predictive utility of YAP where the choice of drug can be used according to the YAP expression pattern. In addition, the protuberant role of YAP makes it an attractive target for the synthesis of anti-cancer drugs.77

Regulating the expression of these genes can be a better approach towards modulating cancer treatment by augmenting the effects of various chemo drugs involved in the whole process. To apply the practice in clinical treatment, the hippo pathway should be extensively studied and can be an accomplishment in targeted therapies for breast cancer treatment.