Cell intrinsic pathways involved in breast cancer plasticity
Cancer cell plasticity is induced by genetic factors and programs which lead to tumor progression, and metastasis to distant organs and confers therapeutic resistance to breast cancer cells. Major intrinsic cell signaling pathways like MAPK, PI3K/AKT/mTOR, Wnt, STAT3, Notch, Hedgehog and TGF-β have been found to be dysregulated in many cancer types and harbor the potential to induce cellular plasticity in cancer cells10.
The MAPK pathway or Mitogen Activated Protein Kinase pathway11 represents a cascade of protein kinases that are considered as the key signaling pathways that regulate cellular and molecular functions like proliferation, apoptosis, differentiation and stress response12. Emergence of MAPK pathway stems back 30 years ago13. Grouped into three families in mammals namely ERKs, p38/SAKs, JNKs 14, this pathway in the canonical form gets activated by an MAPK kinase kinase (MAPKKK). In the case of the ERK pathway of MAPK, Raf is the MAPKK which activates MAPKs MEK 1/2 which in turn mobilizes ERK 1/2. In JNK and p38 pathways of MAPK pathways, TAK1, MEKKs, MLKs and ASK1 act as MAPKKs leading to activation of MKK 4/7 and further downstream pathways involving Jun pathway and also MKK3/6 which activates p3815. Genetic mutation of the MAPK pathway contributes to 58% of tumors with different frequencies of mutations like 42.1% in the JNK pathway, 40.3% in p38, 33.7% in ERK 1/2 pathway and finally followed by only 6.1% in ERK5 pathway 16. Aberrant activation of MAPK pathways and it’s role in cancer progression and development may be due its role in the maintenance of CSCs in tumors. Loss of DUSP4 -dual specificity phosphate-410, a negative regulator of the MAP Kinase superfamily, leads to the development of phenotypes like CSCs in the basal type of breast cancer17. In another study, SIX1which is a developmental homeoprotein activates the MAPK/ERK pathway leading to formation of CSC phenotype in luminal B breast cancer type18. Alongside aberrantly activated MAPK pathway also contributes in promoting EMT which in turn contributes to cancer cell plasticity. It is also associated with different disorders like cancer progression and fibrosis. ERK3 over-expression has also been observed to be associated with increase in cell migration and morphological changes of the breast cancer cells19. Mutations in BRAF gene lead to alterations in the RAS-RAF-MEK-ERK pathway10 which has a major role in developing drug resistance. A pivotal transcriptional factor named FOXC1 is a factor that mediated cancer cell plasticity and metastasis via partial EMT which in turn leads to drug resistance in the same and is regulated by this signaling pathway in breast cancer cells20.
The MAP-kinase pathway has a pivotal role in cellular proliferation in tumors like melanomas21. In the invasive and resistant melanoma cell lines, 229R and 238R, over-activation of MAPK was observed as a result of phenotypic switching. In oral squamous cell carcinoma (OSCC)22, the MAPK pathway plays the role of an intermediary between the epithelial factor GRHL2 and the EMT-inducing signal TGFβ23. In the OSCC cell line SCC9, inhibition of MAPK signaling activated TGFβ signaling which in turn promoted the process of EMT. In the TNBC subtype, MAPK signaling is found to promote the enrichment of breast CSCs24. In SUM-159 and MDA-MB-231 cells, an abrogation of pluripotency factors SOX2, KLF4 and NANOG was seen upon the inhibition of p38-MAPK signaling pathway indicating its role in emergence of breast CSC phenotype. In breast ductal carcinoma, MAPK -ERK pathway was involved in the cytoskeletal reorganization and transition to a mesenchymal phenotype25. Rap1Gap, a GTPase-activating protein26 suppression increased the levels of MAPK/ERK. MCF10A.DCIS cells show higher levels of Rap1Gap when compared to the invasive MCF10A.Ca1D. Rap1Gap silencing in MCF10A. DCIS cells resulted in elevated levels of ERK/MAPK which promoted their invasive capabilities accompanied by skeletal structure re-organization and gain of mesenchymal capabilities further outlining the role of the pathway in phenotypic switching. p44/p42 MAPK activation through Monocyte Chemoattractant Protein-1 (MCP-1)27, which promotes EMT, resulted in an increase in cell invasion28 in TNBC. In Py2T cells, the hyaluronan synthase (HAS2) gene was shown to modulate EMT and cell migration through Erk1/2 MAP-kinase signaling29.
The second pathway that has a major role in cancer cell plasticity is PI3K/AKT/mTOR pathway. The PI3K/AKT/mTOR pathway30also known as the phosphatidylinositol 3 kinase/ protein kinase B pathway, is a highly conserved pathway affecting cellular processes such as proliferation, survival, growth, metabolism and immune response31. The signaling pathway came into prominence in 1977 with the discovery linking PI kinase activity with viral oncogene-encoded Tyr-kinase32. It also has an important role in various oncogenic regulations and thus targeting the PI3K pathway for therapeutic intervention has been a key research area in cancer biology.
In physiological conditions, activation happens by either receptor tyrosine kinases or GPCR (G protein-coupled receptors)33. Activation happens by phosphorylation of the phosphatidylinositol 4,5 biphosphate and converts it to phosphatidylinositol 3,4,5 trisphosphate. This phosphorylated PI3K phosphorylates the kinase domain of AKT at Threonine 308 residue which then binds to various downstream cytoplasmic as well as proteins facilitating the cell survival and growth. The next step to this canonical pathway involves the binding and phosphorylation by AKT to TSC2 which inhibits Rheb. Activated Rheb stimulates the mTOR complex 1 (mTORC1) which effects cell proliferation34. Occurrence of the pathway is observed in both the cytoplasm as well as the nucleus. The cytoplasmic counterpart of the pathway is considered the canonical route for regulating cellular functions. However, PI3K is also present in the nucleus35. Various factors target the pathway and are observed to aberrantly activate the pathway leading to progression of cancer in the patient for example the cancer-testis antigen A-kinase anchor protein 3 (AKAP3)36. In a study by Zhan et al through the use of tissue microarrays and immunohistochemistry, it was observed that AKAP3 was upregulated to a greater extent in cancerous cells of the breast and activates this pathway to facilitate cell migration, progression, migration in breast cancer cells37. This pathway in mediates cellular plasticity which results in the rise of the different cellular populations within the tumor which is documented in several studies like EMT induction that leads to repression of the tight junction proteins which mediates cell plasticity. In breast cancer, hormone receptor-positive tumors have been more associated with mediating cellular plasticity and therapy resistance than breast cancer tumors which occur due to mutation in the PIK3CA gene3. Resistance of TNBC cells towards chemotherapeutic drugs has been observed due to mutation in the PI3K3CA pathway that encodes a subunit of PI3K pathway that causes inhibition of cell apoptosis, also facilitating cell proliferation 38. Another study by Ghebeh et al. which focused on Fascin-mediated chemoresistance in breast cancer elucidated that PI3K/AKT pathway mediates the resistance39. The group used xenograft tumor models to evaluate effect of fascin in mediating chemoresistance in breast cancer cells. This experiment led to significant direct correlation of survival rate of patients who were treated with chemotherapy as a treatment modality and Fascin expression. The increase in chemoresistance of the cancer cells was also attributed to PI3K pathway in a parallel line to increased FAK phosphorylation, expression of XIAP and Livin which are inhibitors of apoptosis proteins, and also proapoptotic marker(caspase 9,3, PARP) suppression.
The PI3K pathway induces EMT directly or through its cooperation with other signaling pathways40 . Activation of the pathway promotes occurrence of EMT during breast cancer progression41 . Mitochondrial ribosomal protein L13 (MRPL13) was found to be a pro-EMT factor, as its depletion resulted in a decrease in mesenchymal markers like SNAI1 and VIM with an increase in the levels of ECAD. Further, it was observed that MRPL13 mediated EMT through PI3K/AKT/mTOR signaling pathway. Interestingly iit was observed that that the PI3K/AKT was involved in conferring the cells’ resistance to Tamoxifen in addition to their role as promoters of EMT in T47D cells42. It was observed that when Connexin (Cx) 43, an EMT inhibitor was silenced alongside reduction in the levels of ECAD as well as an increase of NCAD levels. The addition of LY294002, a specific PI3K/Akt signaling inhibitor43, suppressed EMT, corroborating role of the PI3K signaling in EMT progression. FAK/PI3K/Akt upregulation conferred the cells’ resistance to Cisplatin in addition to promoting EMT44 . Here it was observed that when MDA-M-231 cells and MCF-7 cells here treated with LY294002 it resulted in the attenuation of EMT with decreased levels of mesenchymal factors like SNAI1, SNAI2, VIM and NCAD and increase in the level of ECAD, an epithelial factor. Also, the treatment with LY294002 made cells sensitive to Cisplatin treatment. Analysis using T47D, MDA-MB-231 cell lines demonstrated that the EMT induction by Inorganic pyrophosphatase 1 (PPA1) mediated by the PI3K/AKT signaling pathway45 . When the relationship between KRAS, PI3K/AKT and EMT was analyzed in T47D cells, it was seen that KRAS depletion resulted in the silencing of the signaling factors of PI3K/AKT, alongside EMT inhibition as indicated by the reduced NCAD levels and VIM with increased levels of ECAD46.
STAT3 pathway is another major intrinsic cellular signaling pathway. The Signal Transducers and Activation of Transcription (STAT) 3 part of the STAT transcription factor family has seven highly conserved and structurally similar members. Since its discovery in 1994, STAT3, has been linked with cell growth and apoptosis, as well as in many disorders including different cancer types47. This signaling pathway generally gets activated by growth factors and cytokines leading to creating a docking site for cytoplasmic STAT3. After being activated by phosphorylation, homodimerization takes place from where they are translocated to the nucleus leading to binding to different DNA response elements and regulating their transcription48. This is one of the main intrinsic signaling pathways that in cancer gets aberrantly expressed and plays a major role in cancer cell inflammation. One such role of STAT3 pathway is observed in bladder cancer. In a study by Mirzaei et al, they found hyperactivation of STAT3 led to increased proliferation and metastatic ability of bladder cancer cells49. This is mainly due to the pathway mediating the inhibition of apoptosis and cell cycle arrest which leads to this aggressive behavior of the cancer cells. Another cancer type where STAT3 hyperactivation leads to enhanced cancer cell proliferation, tumor angiogenesis, invasion and metastasis is colorectal cancer50. STAT3 mediated key inflammatory factors and is excessively hyper-activated in colorectal cancer cells. Jung et al showed that STAT3 activated by Low-Density Lipoprotein Cholesterol mediates as well as enhances proliferation of cancer cells of the pancreas and prostate51. It was also observed in cell lines- PANC-1 and LNCaP where STAT3 pathways induced by LDL were deleted reducing the cell proliferation and migration. Similarly, STAT3 and its aberrant expression mediated breast cancer inflammation and proliferation.
The involvement of Wnt pathway in its canonical form is well-established, however more recent research has uncovered the effect of the abnormal functioning of the pathway, through genetic and epigenetic defects, in the initiation, progression and development of drug-resistant characteristics in various cancers, through processes including the modulation of cancer stem cell behavior. The discovery of role of the Wnt pathway in cancer arose as a result of the finding that mammary tumors developing in mice after their infection with the murine mammary tumor virus were often due to the activation of the murine Int-1 gene, which was later named the Wnt-1. Later studies that revealed the relevance of the Wnt pathway in human cancers found that the cytosolic concentrations of β-catenin, a homolog of the Drosophila Armadillo protein with significant roles as the regulator of intracellular adhesion and as transcriptional activator controlled by the signaling pathway, was downregulated by a tumor-suppressor called adenomatous polyposis coli (APC) while the Wnt1 protein upregulated it, providing a basis for the Wnt/ β-catenin pathway-cancer connection. The aberrant functioning of the pathway can be observed amongst different cancers like colorectal, gastric, breast cancers,. Numerous genetically engineered mouse models have revealed that the induction of mammary tumorigenesis occurs as a consequence of the dysregulation of the Wnt/β-catenin pathway via the overexpression of its activators such as Wnt and the targeted disruption or silencing of its inhibitors such as APC.52
In most cancers, (most notably colorectal and gastrointestinal cancers) mutations that result in loss-of-function in the genes coding for the proteins involved in the Wnt pathway that is responsible for the degradation of beta-catenin such as APC and Axin - which are also tumor suppressor genes - have been recognized. As a result, the downregulation of the amount of free cytoplasmic beta-catenin is inhibited and the activation and transcription of the Wnt target genes ensue. In breast cancer, there has been growing evidence indicating the increased expression of pathway activators, suppression of tumor suppressor proteins and the enhanced transcription of Wnt target genes. For example, in transgenic mice with a mutation at the Int-1/Wnt-1 site, the formation of mammary adenocarcinomas with metastasis to the lymph nodes was discovered. Additionally, the mammary tumorigenesis in the same transgenic mice also showed cooperation with various oncogenes coupled with the suppression of several tumor suppressor genes such as the p53 gene. Similarly, in a different mice model carrying the Apc Min/+ allele, which coded for a truncated version of the APC protein, it was discovered that the females carrying the mutation were more likely to develop spontaneous mammary tumors and even showed enhanced sensitivity to mammary carcinogenic compounds. Moreover, various studies have also brought to light that the over-expressed target genes of Wnt/beta-catenin like the c-myc, MMP-3, MMP-7 genes in mammary epithelial cells leads to tumorigenesis. An additional aspect of breast cancer progression where a critical role of Wnt pathway is observed is in the maintenance of plasticity BSCS. Recent research has highlighted that the PCNA-associated factor (PAF), which is seen to control cancer cell stemness, interacts with the Wnt signaling by hyperactivating it via serving as a co-factor of the beta-catenin transcription complex within the nucleus and thereby increasing Wnt target gene expression. This connection was discovered when it was found that PAF expression in mammary epithelial cells upregulated beta-catenin as well as enhanced its translocation into the nucleus53.
Under physiological conditions, the hedgehog (Hh) pathway plays a vital role in embryonic development by patterning the developing neural tube, axial skeleton, limbs, skin, lungs, hair and teeth. However, the contribution of the aberrant form of the pathway to tumorigenesis is being made clear by accumulating evidence in recent years. However, the first clue connecting the hedgehog signaling pathway to cancer came as a result of the discovery that a mutation in the tumor suppressor gene that encodes for a receptor molecule found within the pathway called patched (Ptc) was an underlying factor in the development of both familial and sporadic forms of basal cell carcinoma (BCC).
The Notch pathway is a tightly controlled ssignaling pathway responsible for various aspects of development of metazon as well as renewal of tissue. As a conserved signaling pathway, the Notch pathway plays a critical role in short-range communication between cells through the establishing physical contact between the target cells. The pathway is responsible for enabling and suppressing, depending on context and molecular cues, and cellular functioning like cell differentiation, proliferation of cells, cell death and controlling cell fates throughout the development of the organism as well as in self-renewing adult tissue. Given the vital role it plays, the mis-regulated loss-of or gain-of function of the Notch pathway can give rise to various human diseases, including cancer. Given the fundamental role played by Notch in determination of cell fate and the maintenance of adult stem cells and progenitor cells, its dysregulation has been linked to the maintenance of cancer stem cells (CSC) which, as growing evidence suggests, could be the initiator of tumorigenesis, according to the cancer stem cell model.  In breast cancer specifically, studies have shown that Notch acts as an oncogene. The overexpression of the Notch proteins 1, 3 or 4 has been shown to transform normal breast epithelial cells to cancer cells, leading to the overactivation of the signaling pathway.