Cancer cell genotypes are a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth; self-sufficiency in growth signals, insensitivity to growth-inhibitory (anti-growth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis. Environmental and endogenous DNA-damaging agents and genetic instability drive tumor progression by generating mutations in two types of genes, oncogenes and tumor suppressor genes, providing cancer cells with selective growth advantage and thereby leading to the clonal out growth of a tumor. In general, oncogenes (called proto-oncogenes in their normal, non-mutated form) promote cell proliferation and survival, whereas tumor suppressor genes inhibit cell growth. Cells proliferate only when required, as a result of delicate balance between growth promoting and growth-inhibiting mechanisms that are controlled by an intricate network of intra- and extracellular molecules. In stark contrast, cancer cells override these controlling mechanisms and follow their own internal program for timing their reproduction (Ref.1). The major human oncogenes encoding oncoproteins are like Ras (point mutation); MIZ1, Myc and MAX (point mutation, amplification); c-Jun and c-Fos (increased expression); CcnD (amplification, translocation); CcnE (amplification); CDK2 (amplification, increased expression); CDK4 (point mutation); FkhR/FoxO1 (translocation); HPV-E6 and HPV-E7 (viral infection); MDM2 (amplification); RAR (Retinoic Acid Receptor); SOX (amplification, increased expression); Abl (translocation); Gli (amplification, translocation); Ctnn-Beta (point mutation); Aurora-A (amplification, increased expression); CDK4 (point mutation); DCR and FLIP (mutation); BAX (point mutation); Akt (point mutation, amplification, increased expression); PI3K (point mutation); JAK (point mutation, translocation); RTKs (point mutation, translocation, amplification, increased expression); Src, Crk, Cbl, and Fyn (mutation); Rho and Rac (increased expression); BRaf (point mutation); GPCR and GN-Alpha (point mutation); Hh and Smo (point mutation); Notch (translocation); and WNT1 (increased expression). Whereas the major tumor suppressor proteins include TGF-BetaR1/R2 (point mutation); BMPR1/R2 (point mutation); SMAD2/3/4/5 (point mutation); p16(INK4A) or p14(ARF) (point mutation); p15(INK4B) (point mutation); p27(KIP1) (mutation); p21(CIP1) (mutation); Rb (point mutation); p53 (point mutation; deletion); Chk1/Chk2 (point mutation); ATM and ATR (point mutation); BRCA1 (point mutation); NBS1 (point mutation; deletion); p18(INK4C) (mutation); p19(INK4D) (mutation); FANCD2 (point mutation); DNA-PK (point mutation); HIPK2 (point mutation); VHL (point mutation); NF1 (point mutation; deletion); Bcl2 (translocation); PTEN (point mutation, deletion); Integrins (deletion); SuFu and Ptc (point mutation); WNT5A, AXIN, APC, CdhE and Ctnn-Alpha (point mutation). This review focuses on key signaling routes (GPCR Signaling; Ras/Integrin Signaling; Akt/RTKs/Cytokine Receptor/NF-KappaB Signaling; TGF-Beta/BMP Signaling; WNT Signaling; Notch Pathway; Hedgehog Signaling and Death Receptor Signaling) participating in intercellular and intracellular communication whose deregulation by the above mentioned oncoproteins and tumor suppressors leads to the acquisition of the malignant phenotypes that are over-expressed in many human cancers (Ref.1 & 2).
Within a normal tissue, multiple anti-proliferative signals operate to maintain cellular quiescence and tissue homeostasis. Incipient cancer cells evade these anti-proliferative signals in order to prosper. Cancer cells also switch to different types, favoring only the ones that transmit pro-growth signals. The WNT Signaling plays a central role in the development of many phylogenetically diverse organisms. Genetic alterations of components in the WNT pathway are commonly associated with melanomas and carcinomas of the breast and colon including the FAP (Familial Adenomatous Polyposis) syndrome. In the canonical WNT pathway GSK3Beta mediated Ctnn-Beta ubiquitination and degradation are inhibited by the binding of WNT1 (includes WNT1, WNT2, WNT3A, WNT8) to the receptor-coreceptor complex Fzd and LRP5/6. However WNT5A class (including WNT5A, WNT4, WNT11) inhibits binding of WNT1 to Fzd and LRPs. They ultimately lead to the activation Dsh that in turn inhibits GSK3Beta function. In the absence of WNT, Ctnn-Beta forms multiprotein complexes with Axin and APC, leading to its phosphorylation and degradation. With WNT stimulation, GSK3Beta activity is down modulated and unphosphorylated Ctnn-Beta then accumulates and translocates to the nucleus, to signal as a heterodimer with LEF/TCF proteins (Ref.3). The TCFs which bind and dimerize with Ctnn-Beta, along with LEF1, are TCF3 and TCF4. The “armadillo” repeats of free Ctnn-Beta also interacts with CdhE, Src (colon cancer), Ctnn-Alpha, p120Ctn and CBP. CdhE function is apparently lost in a majority of epithelial cancers. The disruption of CdhE/Ctnn-Beta-mediated gene transcription is caused by Cd2+ (Cadmium), SOX (Blepharophimosis, Ptosis, Epicanthus Inversus Syndrome, Moebius Syndrome, myeloid leukemia, Acampomelic Campomelic Dysostosis, primary colorectal tumors, primary breast cancer, primary kidney tumors, and primary lung and prostrate cancer) and RAR (hepatocellular carcinoma) which further stimulates the development of tumors. Ctnn-Beta mutations are a crucial step in the progression of ovarian, hepatocellular, endometrial and prostrate cancers (Ref.3 & 4).
Several other genes not directly involved in WNT signaling but have overlapping functions with the canonical WNT pathway. Many of these genes are components of the Hedgehog pathway. The Hh-signaling comprises three main components: the Hh ligand; a transmembrane receptor circuit composed of the negative regulator Ptc, plus an activator, Smo a GPCR (G-Protein Coupled Receptor); and finally a cytoplasmic complex that regulates the Gli family of transcriptional effectors. Ptc, a twelve-pass membrane protein binds Hh ligand, and in the absence of ligand, Ptc interacts with and inhibits Smo. This repression culminates in a transcription factor, Gli acting as a transcriptional repressor. When Hh binds Ptc, its interactions with Smo are altered such that Smo is no longer inhibited. This leads to Gli protein entering the nucleus and acting as a transcriptional activator for the same genes it represses when Ptc is free to interact with and inhibit Smo. The processing and nuclear import of Gli is regulated via a complex of Gli with the cytoplasmic members of the Hh pathway, Fused (Fu) and SuFu (Suppressor of Fused). On Hh signaling, the complex is released from microtubules
and full-length Gli enters the nucleus. In the absence of Hh signal, Fu and SuFu binding to Gli prevent Gli activation and retain it in the cytoplasm. Upon Hh reception, Fu is activated and acts on SuFu, alleviating their negative effect on Gli. As a result, Gli cleavage is reduced, Gli nuclear import overcomes its export and Gli is activated. Gli activation requires Fu to antagonize SuFu negative effect. Activated nuclear Gli is fully involved in the transcription of Hh target genes, like WNT, Ptc and BMPs. Mutations in the components of the Hedgehog pathway like Ptc, are responsible for the inherited cancer predisposition disorder known as Gorlin or Nevoid Basal Cell Carcinoma Syndrome (NBCCS), medulloblastoma, squamous cell carcinomas of the esophagus, transitional cell carcinomas of the bladder, and the benign skin lesions, trichoepitheliomas (Ref.4 & 5). However WNT signaling components like Dsh directly modulate the Notch signaling. The Notch COOH-terminal fragment NEXT is cleaved by Gamma-Secretase (Presenilin, PEN2, APH1 and Nicastrin) and TACE to release NICD into the cytoplasm. Upon release, the NICD translocates to the nucleus and associates with the RBPJ-Kappa, p300, HAT1 and HIF1Alpha which activate the expression of a set of target genes, including the HEY1/2, HES1 among others. This translocation is opposed by Numb and Dsh. HIF1Alpha gene regulation results in VEGF, Epo and PDGF expression and alterations in HIF1Alpha gene leads to sustained Angiogenesis for which it is often inhibited by different cellular levels of VHL (renal cell carcinoma, cerebellar hemangioblastoma). Consistent with the ability to influence cellular differentiation in multiple tissues, mutations of Notch receptors and components of its signaling pathway have been associated with a number of diseases, including human T-cell leukemia, CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), Spondylocostal Dysostosis, Alagille syndrome and breast carcinomas (Ref.6).
Another prominent signal transduction pathway like the versatile WNT signaling is the TGF-Beta superfamily of growth factors that are divided into two general branches; the TGF/Activin/Nodal and BMP/GDF branches. These branches propagate signals by phosphorylating the SMAD family of intracellular mediators at their carboxyl-terminal ends. SMADs are divided into three distinct classes; receptor regulated R-SMADs (SMAD1, 2, 3, 5 and 8); co-SMAD (SMAD4) and inhibitory SMADs (SMAD6 and SMAD7). The inhibitory SMADs antagonize TGF-Beta signaling. R-SMADs complexes like SMAD1, 2, 3, 4 and 5 are translocated to the nucleus by BMPs>BMPR1/R2; TGF-Beta>TGF-BetaR1 and TGF-Beta> TGF-BetaR1/R2> Ras>ERK signaling routes, where they associate with other molecules like p107, E2Fs, MIZ1, Myc and MAX. The TGF-Beta> TGF-BetaR1/R2 >TAK1 >TAB1>MKK3/6>p38 and TGF-Beta> TGF-BetaR1/R2 >TAK1>TAB2>MKK7 regulates transcription of Myc/MAX and c-Jun/c-Fos, respectively. On the other hand TGF-Beta negatively regulates WNT signaling by inhibiting LEF1/TCF2-3 function through the BMPs>BMPR1/R2>TAK1>TAB1/2>NLK signaling route. Indeed nearly every component of the TGF-Beta pathway is altered in cancer. The main targets appear to be TGF-BetaRs (gastrointestinal cancers (including esophageal, gastric, hepatocellular and colorectal cancers), cervical carcinoma, lymphoma and breast cancer metastases); SMADs (pancreatic and colorectal cancers); whereas oncogenic Ras under the influence of TGF-Beta is associated with the development of aneuploidy and malignant transformation (Ref.7 & 8). The growth inhibitory effects of TGF-Beta are frequently mediated through the induction of Cyclin dependent kinase inhibitors (CKIs) like p15(INK4B); p21(CIP1); p27(KIP1) and down regulation of CDKs and Cyclins like CcnD, CcnE, CcnA, CDK2, CDK4 and CDK6. These along with other CKIs like p16(INK4A), p18(INK4C), p19(INK4D) and viral oncoprotein HPV-E7 are critical for the regulated progression through the cell cycle and therefore maintain the retinoblastoma protein, Rb in the hypophosphorylated state, which prevents the transcription factor E2F from inducing the aberrant expression of genes required for G1-S phase transistion (Ref.9 & 10).
Like p16(INK4A), ARFs (Alternative reading Frames), or transcript variants of p16(INK4A) encodes p14(ARF), a tumor suppressor but its action is distinct from that of p16(INK4A). p16(INK4A) acts on the Rb pathway by inhibiting CDK4/6 and CcnD, whereas, p14(ARF) acts on the p53 pathway by interacting with MDM2, thereby blocking degradation of p53 and resulting in p53 stabilization and activation. Furthermore, binding of p14(ARF) to MDM2 results in sequestration of MDM2 in the nucleolus. This prevents shuttling of MDM2 between nucleus and cytoplasm (to where it must export p53 for degradation), thereby leaving p53 free within the nucleus to regulate the transcription of p53 responsive genes. The p21(CIP1) is also up regulated by the tumor suppressor protein p53, inhibiting CDK2, CDC2, CcnB and CcnD (Ref.9). UV- and IR-induced DNA damage, a major cause for human carcinomas, activates ATM and ATR (both promote Ataxia Telangiectasia), DNA-PK and HIPK2. Notably, phosphorylation at Ser15 by ATM/ATR, either directly or through Chk1/Chk2 or at Ser20 by Chk1/Chk2 has been shown to alleviate the inhibition or degradation of p53, leading to p53 stabilization and activation. Oncogenic Aurora-A (breast, ovarian, colon, prostate, neuroblastoma and cervical cancer) is critical for up-regulation of p53 activity, whereas is HPV-E6 inhibits the same. The key substrates of Chk1/Chk2 include CDC25A, CDC25B and CDC25C. CDC25A activates CDK2/CcnE and CDK4/CDK6/CcnD to promote progression through S phase; while CDC25B/C activates CDC2/CcnB and promotes progression from G2 into mitosis by inhibiting the action of Wee1 (it inhibits CDC2 function by phosphorylating it on Tyr15. ATM/ATR induced signaling also activates Abl, BRCA1, FANCD2, NBS1 to promote DNA repair by modulating the function of downstream elements like Rad51, Rad52 and Chk1 (Ref.11). Aberrant expression of CKIs, CDKs and Cyclins leads to establishment of cancers of breast, prostate, gastric, bladder, esophagus, colon, ovary, nasopharynx and pancreas; cervical carcinoma; hepatocellular carcinoma; oral squamous cell carcinoma; thyroid carcinoma; squamous cell carcinoma of head and neck; cutaneous malignant melanoma; acute lymphoblastic leukemia; primary cutaneous B-cell lymphoma; granulosa cell tumor; myelopoiesis; chronic lymphocytic leukemia; parathyroid adenoma; Wilms Tumor and Denys-Drash Syndrome, etc to name a few. Similarly mutations in Rb is associated with retinoblastoma, bladder cancer and small cell cancer of the lung; whereas, p53 mutations are responsible for rhabdomyosarcoma, hepatocellular carcinoma, colorectal cancer, lung cancer, brain tumor, esophageal carcinoma, bladder cancer, squamous cell carcinoma, cervical and anal cancer; breast cancer and Li-Fraumeni Syndrome (Ref.12). Oncogenic Abl contributes to chronic myeloid leukemia, acute lymphoblastic leukemia and acute myelogenous leukemia and its activation controls signaling of two main oncoproteins; Ras and Akt. In many cases Ras signaling is inhibited by the tumor suppressor NF1 (Neurofibromatosis, Watson syndrome, Juvenile Myelomonocytic Leukemia). Ras and Akt are generally mutated during the development of breast cancer, urinary tract tumors, norwegian lung cancer, bladder cancer, prostate cancer, pancreatic cancer, endometrial tumorigenesis, liver angiosarcoma, colorectal cancer, stomach cancer, acute myelogenous leukemia, multiple myeloma and primary plasma cell leukemia, acute myeloid leukemia, leiomyosarcoma and ovarian carcinoma (Ref.11 & 13).
Akt and Ras are regulated via major signaling routes like GPCR signaling, Ras signaling, Integrin signaling, Akt/RTKs/Cytokine Receptor/NF-KappaB signaling and Death Receptor signaling among others, that control cell growth through activation and/or deactivation of cell cycle arrest and apoptosis. Oncogenic GPCR/GN-Alpha (implicated in neuroendocrine tumors, prostate cancer, neuroblastoma, pancreatic cancer, Lung cancer, liver cancer, etc to mention a few) interact with other cognate G-proteins like G-Beta and GN-Gamma enhances Cyclin expression and cell proliferation. Under GPCR signaling Ras is activated by GN-Alpha/Beta/Gamma> PLC-Beta > IP3> CamK2>RasGRF; GN-Alpha/Beta/Gamma>PLC-Beta>IP3>CamK2>RasGRP; GN-Alpha/Beta/Gamma> PLC-Beta >DAG>PKC>RasGRP; GN-Alpha>PI3K-Gamma>RasGEF and GN-Alpha/Beta/Gamma> PLC-Beta >DAG>PKC signaling routes with GN-Alpha/Beta/Gamma> PLC-Beta>IP3>CamK2>SynGAP inhibiting the same. PKC enhances Ras function by inhibiting RasGAP function. Further Ras>c-Raf>MEK1/2-MP1-ERK1/2 activates c-Jun/c-Fos to regulate Cyclin expression. Ras enhance cell proliferation mediating Akt signaling through PI3K/PIP3 activation, with GN-Alpha>PI3K-Gamma>PIP3 being the main route for GPCR-induced Akt activation. PI3K is up-regulated in various cancer types that include breast cancer, ovarian cancer, colorectal cancer, anaplastic thyroid carcinomas. The GN-Alpha> > cAMP>PKA/EPAC1 route activating oncocogenic BRaf (up-regulated in lung adenocarcinoma, melanocytic neoplasia, colorectal cancer) links hormone-induced GPCR signaling with Growth Factor stimulated RTKs (GRB2/SOS>Ras; Src>Ras; Crk>C3G>Rap). BRaf activation is also enhanced by ECM/Integrin interactions (GRB2/SOS>Ras; Fyn/SHC>Ras) (Ref.14 & 15). RTKs mostly deregulated in myofibroblastic tumor, anaplastic large cell lymphoma, thyroid tumorigenesis, small cell lung cancer, prostate cancer, acute myeloid leukemia, breast cancer, colon cancer, epithelial tumor, gastric cancer, multiple myeloma, colorectal cancer, pituitary tumor, astrocytomas, Ovarian Carcinoma, hepatocellular carcinoma, lymphoblastic leukemia, gall bladder carcinoma, gastrointestinal stromal tumor, germ cell tumor, medullary thyroid carcinoma, fibrosarcoma, medulloblastoma, basal cell carcinoma, chronic myelomonocytic leukemia, multiple endocrine neoplasia and multiple venous malformations in skin and mucous membranes, where as aberrant expression of Integrins leads to schwannomas, breast cancer, Kaposis sarcoma, small cell lung cancer, colon cancer, bladder cancer and melanomas. RTKs and ECM/Integrin induction of BRaf and Ras controls cell cycle regulation by activating c-Jun/c-Fos via Ras> BRaf> MEK1/2/ MP1/ ERK1/2>Elk; Ras> BRaf> MEK1/2/ MP1/ ERK1/2>Elk >JNK; Ras> RalGEF> RalA/B>RalGAP>Rac>JNK; Ras>RalGEF>JNK; Ras>Rac>JNK; Ras/Integrin>Rho>CDC42>PI3K>Rac> JNK; Integrin>CDC42/FAK >PI3K>Rac>JNK signaling routes. Rho and Rac are mutated mostly in benign, malignant and inflammatory breast cancer; Non-Hodgkins lymphoma and ovarian cancer. Up-regulation in c-Jun and c-Fos expression is a significant characteristic feature of breast cancer, hepatocellular carcinoma, fibrous dysplasia and osteosarcoma (Ref.16 & 17).
Growth Factor/RTKs signaling also couples with Cytokine/Cytokine Receptor signaling to regulate Akt signaling by activating the common downstream target PI3K. Cytokines and their receptors recruit Shp2, GRB2, SOS, Ras, SHC and JAKs (breast cancer, head and neck cancer, multiple myeloma, erythroleukemia, acute myelocytic leukemia, lymphomas) to activate PI3K, whereas Growth Factors and RTKs recruit GAB1/2, Cbl and IRS1 to activate PI3K. PI3Ks
increase PIP3 levels to activate Akt directly or through PDK-1 actvation. PIP3 accumulation in the cytosol is checked by the tumor suppressor PTEN (common in Cowden disease, cervical cancer, Bannayan-Zonana syndrome, Lhermitte-Duclos disease, endometrial carcinoma, prostate cancer, malignant melanoma, oligodendroglioma and glioblastomas) (Ref.18). At the molecular level, all anti-proliferative signals are funneled through Akt that controls the mitochondrial and Fas/FasL (squamous cell carcinoma, colon carcinoma, Hashimotos thyroiditis) mediated apoptosis, energy depletion pathways, NF-KappaB signaling among others. The Fas receptor upon binding to the FasL trimerizes and induces apoptosis through a cytoplasmic domain called DD (Death Domain) that interacts with signaling adaptors like FADD, TRADD and ASK1 to recruit Caspase8 and 10. Upon activation, Caspase8 and 10 cleave and activate downstream effector Caspases, including Caspase3, Caspase6 and Caspase7. Activated Caspase8 activates Caspase3 through two pathways; the complex one is that Caspase8 cleaves BID and another pathway is that Caspase8 cleaves Procaspase3, 6 and 7 directly and activates it. Both pathways are regulated at the level of Caspase8 activation by the endogenous inhibitor FLIP and DCR/DecoyR (both are involved in ovarian carcinoma, squamous cell carcinoma, colon carcinoma, Hashimotos thyroiditis). BID enters the mitochondrial apoptotic pathway as tBID (truncated BID) where it facilitates MMP (Mitochondrial Membrane Permeabilization) through BAX (uterine leiomyomas, acute lymphoblastic leukemia) and BAK activation along with BIM. However, this mechamism is counterbalanced by BclXL (Non-Hodgkins lymphoma) and Bcl2 (follicular lymphoma) activity. MMP triggers the release of mitochondrial pro-apoptotic factors like CytoC and SMAC. The released CytoC binds to APAF1/Caspase9. Caspase9 in turn activates Caspase3 and SMAC represses anti-apoptotic proteins like CIAP to promote apoptosis. Incipient cancer cells evade apoptosis to proliferate at random. For which Akt inhibits BAD directly or through PAK1>c-Raf activity; inhibits ASK1; and activates MDM2 to inhibit p53. p53 interferes with apoptosis by activating Noxa and PUMA, both being negative regulators of Bcl2. Akt also regulates cell cycle by inhibiting the action of GSK3 (thereby CcnD) and FoxO1 (elevated during alveolar rhabdomyosarcoma). Energy depletion leads to the activation of LKB1>AMPK>TSC1/2. TSC1/2 is vital for mTOR Pathway that controls autophagy and translation by inhibiting Rheb>mTORC1 complex signaling cascade. But Akt inhibit TSC1/2 function as their mutations lead to pulmonary lymphangioleiomyomatosis and tuberous sclerosis (Ref.19 & 20). In general, the recent elucidation of the molecular mechanisms by which normal cells transduce proliferative signals and grow or make life or death decisions; provides a golden opportunity in the search of novel molecular targets for pharmacologic intervention in cancer. It has also been revealed that the change of miRNA expressions contributes to the initiation and progression of cancer and more than fifty percent of miRNAs are located in cancer-associated genomic regions or in fragile sites. This shows the possibility that miRNAs have clinical benefits as not only therapeutic targets but also a tool for cancer diagnosis (Ref.2).