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.