Cancer cell signaling

1. Notch signaling pathway is highly conserved molecular cell signaling pathway that regulates a
vital role in proliferation, stem cell maintenance, cell fate specification, differentiation, and
homeostasis of multicellular organism and implicates angiogenesis. Mammals possess four
different notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The notch
receptor is a single-pass transmembrane receptor protein. It is a hetero-oligomer composed of a
large extracellular portion, which associates in a calcium-dependent, non-covalent interaction with
a smaller piece of the notch protein composed of a short extracellular region, a single
transmembrane-pass, and a small intracellular region.
Notch Signaling Pathway

2. Notch signaling is initiated by ligand binding to Notch receptor, which undergoes a two-step proteolytic
cleavage by ADAM family proteases and γ-secretase, releasing the Notch intracellular domain (NICD).
The NICD translocates to the nucleus where it binds to CSL and converts the complex from a repressor to
an activator of Notch target genes.
Notch absence:
Transcription repressor
Notch present:
Transcription activator

3. Crosstalk signaling pathway
Crosstalk refers to instances in which one or more components of one signal transduction pathway affects
another. In these signal transduction pathways, there are often shared components that can interact with
either pathway.
For example, YAP/TAZ and Notch interplay that impacts on the balance between stem cells’ self-renewal
versus differentiation, cell fate decisions, inflammation, morphogenesis, and large-scale gene oscillations.

4. Figure: Yap/Taz forms a
critical positive feedback
loop with Notch
signaling, to promote
liver enlargement and
tumorigenesis (Red).
Breaking this positive
feedback loop leads to
reduced hepatomegaly
and tumor progression.
The inhibitory role of
Wnt/β-catenin in the
liver tumor caused by the
vicious positive feedback,
is at least in part through
the DP1 (Dimerization
Partner of E2F
transcription factor)-
mediated inhibition of
Notch signaling (Blue).

5. Notch Inhibitors
Notch signaling could be targeted by inhibiting the signaling pathway by two major classes of notch inhibitors that
primarily focuses on the clinical development of promising agents that either obstruct Notch receptor cleavages
such as γ-secretase inhibitors (GSIs) or interfere with the Notch ligand-receptor interaction by monoclonal
antibodies (mAbs).
Combining notch inhibitors with current cancer therapies can be effective method for treatment strategies that can
give the most promising results.

6. γ-secretase is a large protease complex and is composed of catalytic and accessory subunits. The activation of Notch
signaling pathway mainly depends on the γ-secretase enzyme activity that helps in the proteolytic cleavage of the
receptors that release the active intracellular fragment which is one of the most crucial steps. Thus, GSIs is a
promising target for Notch inhibition. GSI were the first class of inhibitors that reached clinical development in
oncology. There are more than 100 GSIs synthesized and they can be divided into three classes:
• Peptide isosteres
• Azepines
• Sulfonamides
GSI

7. Name Target Type of study
MK-0752 (Merck and Co,
Whitehouse Station, NJ, USA)
Metastatic or locally advanced
breast cancer
Phase I
PF03084014 (Pfizer, Groton,
CT, USA)
Advanced solid tumors Phase I
RO-4929097 (Roche, Nutley,
NJ, USA)
Advanced or metastatic breast
cancer or recurrent triple
negative breast cancer
Phase II
Clinical trials employing γ-secretase inhibitors in the treatment of breast cancer:

8. Monoclonal antibodies (mAb)
These are mAbs tested in clinical trials, an example being MAb604.107, which recognize specific ligand
(DLL-4) or receptors (Notch1–3) and they either prevent ligand/ receptor interaction or the
conformational change within the extracellular domain which is required to expose the TACE
cleavage site. Delta like ligand 4 (DLL-4) is an important component of NOTCH pathway that controls
the proper growth, stem cell renewal and development.

9. AKT signaling pathway (The survival pathway)
AKT is a serine/threonine kinase, also known as protein kinase B (PKB).
Akt signaling pathway has roles in-
• Cell cycle progression
• Regulation of glucose metabolism (for the generation of new biomass and facilitate nutrient signaling)
• Protein synthesis
• Promoting cell survival by blocking apoptosis

10. AKT has three isoforms:
• AKT1, AKT2, AKT3
Structure:
Consists of three domains:
1. N-terminal domain: PH domain (Pleckstrin homology-domain)
consisting of 100 amino acid, which interacts with PIP3
(phosphatidylinositol triphosphate) and PIP2 (phosphatidylinositol
bisphosphate).
2. Central domain: kinase domain having a regulatory threonine
residue, Thr308.
3. C-terminal domain: consists of 40 amino acids having a regulatory
serine residue, Ser473.
Figure: Isoforms of AKT

11. Activation of AKT signaling
Protein
synthesis
autophagy
Glycogen
synthesis
PIP2

12. • AS160: Negative regulator of GLUT 4 translocation
• Rheb: Activator of mTORC1 pathway
• TSC 1/2: Inhibitor of mTORC1 pathway
• FOXO: Inhibitor of cell survival and proliferation
• GS (Glycogen synthase): Helps glycogenesis
• GSK (Glycogen synthase kinase): Phosphorylates glycogen synthase (GS) to inactivate it
In a nutshell, an activated AKT signaling pathway-
 Increases glucose uptake and utilization by cells
 Enhances glycogen synthesis
 Increases fatty acid synthesis
 Increases protein synthesis
 Upregulates cell survival and proliferation
 Downregulates autophagy

13. Inactivation of AKT signaling
Glycogen
synthesis
autophagy
Protein
synthesis

14. Crosstalk between signaling pathways

15. AKT in Cancer
AKT1 gene amplification has been reported in gastric carcinoma, glioblastomas, and gliosarcomas.
AKT2 gene amplification has been identified in head and neck squamous cell carcinoma, pancreatic, ovarian, and
breast cancers.
AKT3 expression in androgen resistant prostate cancer cell, estrogen receptor deficient breast cancer cells and in
primary ovarian cells.
• Mutations leading to the amplification of genes in the receptor-PI3K pathway, resulting in enhanced PI3K
signaling.
• PTEN activity can be impaired by various mechanisms like, somatic mutations, homozygous deletion, epigenetic
silencing through gene promoter methylation or post transcriptional modifications.
• The AKT activated mTOR signaling pathway negatively regulates autophagy.

16. Overexpression of AKT is linked to resistance to chemotherapeutic agents such as cisplatin, methotrexate or
paclitaxel.
• Cisplatin-induced DNA damage causes the phosphorylation of BAD via AKT, suppressing its (BAD) apoptotic
effect.
• However, MK-2206, an AKT inhibitor, has shown to improve the effectiveness of cisplatin in gastric cancer cell
lines and ovarian cancer cell lines.

17. AKT Inhibitors
1. ATP competitive agents: GSK690693, afuresertib, uprosertib, AZD5363, ipatasertib etc.
Resistance of ATP competitive inhibitors: ATP competitive inhibitor A-443654 induce “paradoxical” AKT hyper-
phosphorylation. Targeting AKT kinase to the cell membrane markedly reduce sensitivity of phosphorylated AKT to
dephosphorylation by protein phosphatase 2A (PP2A). This effect was amplified by occupancy of the ATP binding
pocket by either ATP or ATP-competitive inhibitors. Thus, occupancy of the nucleotide binding pocket of AKT
kinases enables intramolecular interactions that restrict phosphatase access and sustain AKT phosphorylation.
2. Allosteric inhibitors: Allosteric modulators offer distinct advantages compared to orthosteric ligands that target to
active sites, such as greater specificity, reduced side-effects and lower toxicity. e.g. MK-2206, Triciribine, BAY
1125976 etc.

18. Class Description
ATP-competitive inhibitors Orthosteric inhibitors targeting the ATP-binding
pocket of the protein kinase B (Akt)
Isoquinoline-5-sulfonamides H-8, H-89, NL-71-101
Azepane derivatives Structures derived from (−)-balanol
Aminofurazans GSK690693
Heterocyclic rings 7-azaindole, 6-phenylpurine derivatives, 3-
aminopyrrolidine, AZD5363, ipatasertib, A-674563, A-
443654
Phenylpyrazole derivatives AT7867, AT13148
Thiophenecarboxamide derivatives Afuresertib, uprosertib
Allosteric inhibitors Superior to orthosteric inhibitors providing greater
specificity, reduced side-effects and less toxicity
2,3-diphenylquinoxaline analogues MK-2206
Alkylphospholipids Edelfosine, ilmofosine, miltefosine, perifosine, erufosine
AKT- inhibiting drugs listed into major classes:

19. Class (cont.) Description
Indole-3-carbinol analogues Indole-3-carbinol, 3-chloroacetylindole,
SR13668, OSU-A9
Sulfonamide derivatives PH-316, PHT-427
Thiourea derivatives PIT-1, PIT-2, DM-PIT-1
Purine derivatives Triciribine, triciribine mono-phosphate active
analogue (TCN-P), ARQ 092
Other structures, derivatives BAY 1125976, 3-methyl-xanthine, 3α- and 3β-
acetoxy-tirucallic acids, acetoxy-tirucallic acid
Irreversible inhibitors Natural products, antibiotics
Lactoquinomycin, Frenolicin B, kalafungin,
medermycin

20. MAPK Pathway
• The MAPK/ERK pathway (also known as Ras-Raf-MEK-ERK pathway) is a chain of proteins in the cell that
communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell.
• The pathway includes many proteins including MAPK (mitogen-activated protein kinases, originally called
ERK, extracellular signal-regulated kinases), which communicate by adding phosphate groups to neighboring
protein which acts as an “on” or “off” switch.

21. Understanding the MAPK pathway as it relates to oncology
• MAPK pathway plays a role in the regulation of gene expression, cellular growth and survival.
• Abnormal MAPK signaling may lead to increased or uncontrolled cell proliferation and resistance to
apoptosis.
• Research into the MAPK pathway has shown it to be important in some cancers such as- lung cancer,
colorectal cancer, pancreatic cancer, endometrial cancer etc.
• The MAPK pathway includes the signaling molecules Ras, Raf, MEK, and ERK.
• Normally extracellular growth factors activate the pathway by binding to receptor tyrosine kinases. This
mobilizes a cascade of signaling via the MAPK pathway signaling molecules.
• Ultimately, activation of the MAPK pathway leads to the transcription of genes that encode proteins involved
in the regulation of essential cellular functions such as cell growth, cell proliferation and cell differentiation.

22. Understanding the MAPK pathway as it relates to oncology (Cont.)

23. MAPK pathway as tumor suppressor
P38 pathways regulate Ras
oncogene activity by negative
feedback (A) and signaling
integration (B).
Along the P38 pathway, MKK6,
P38α, and MK2/PRAK have been
shown to be activated by Ras
oncogene through the MEK/ERK
pathways and in turn, suppress Ras
activity by negative feedback (A).
The P38γ expression, on the other
hand, is transduced by Ras, that is
required for Ras transformation.
Phosphorylated P38α is shown to
downregulate P38γ protein
expression by ubiquitin/proteasome
pathways.
In a given system, Ras transforming activity will be determined by integrated signaling from Ras-suppressor P38α and
the Ras-effector P38γ (B).

24. Cross talk with other signaling pathways
• MAPK pathway and TGF-β:
One of the stimulating factors of MAPK pathway is Ras protein which can antagonize TGF-β induced apoptosis
and cell cycle arrest, resulting in cancer cell proliferation. The synergistic relation between these two pathways
lead to increased cytokines and growth factors and thereby epithelial to mesenchymal transition (EMT).

25. Cross talking with other pathways

26. Drugs against MAPK
Figure: Imatinib, Nilotinib, Dasatinib, and Sunitinib target and inhibit c-KIT. Selumetinib and Trametinib inhibit
MEK activity. Temsirolimus and Everolimus inhibit the mTOR protein. Resistance to Vemurafenib arises from
MAPK pathway reactivation by (1) a MEK1C121S mutation, (2) NRASQ61R/K mutations, (3) COT1
overexpression, (4) alternatively spliced variants of BRAFV600E or amplification of the mutant BRAF allele, (5)
Overexpression or activation of RTKs (PDGFRβ or IGF1R) that bypasses mutant BRAF and activates ERK via
CRAF-MEK or through independent ERK mechanisms by activating the PI3K/AKT pathway.

27. Transforming growth factor-β (TGF-β) signaling pathway
TGF-β cell signaling pathway has major roles in-
 Cell proliferation
 Cell survival
 Apoptosis
TGF-β ligand superfamily includes:
 Activin
 Nodal
 TGF-β
 Bone morphogenetic proteins etc.

28. TGFβ-2 signaling pathway
1. TGF-β ligand binds to TGFβ-2 receptors which are serine/ threonine receptor kinases.
2. TGFβ-2 then phosphorylates serine residue of type-1 receptor and forms hetero-tetrameric complex.
3. SARA (SMAD anchor for receptor activation protein) internalizes the complex and permits binding of R-
SMADs (SMAD2, SMAD3) to L45 region of the type 1 receptor.
4. SARA orients R-SMADs in such a way that R-SMADs’ C-terminus faces with type 1 receptor.
5. Type 1 receptor phosphorylates serine residue of R-SMADs.
6. Phosphorylation of R-SMADs causes their dissociation from SARA.

29. TGFβ-2 signaling pathway (Cont.)
7. SMAD 2/3 form complex with coSMAD (SMAD 4).
8. The complex then enter into nucleus and binds with transcription factor and starts transcription of DNA.

30. TGFβ signaling pathway

31. How TGF-β is associated with cancer
● TGF-β has dual role in cancer.
● It plays both as tumor suppressor and oncogene.
 Tumor suppressive role is often lost by mutation in TGFβ or SMAD pathways such as in colorectal cancer, breast
cancer, gastrointestinal cancer, pancreatic cancer etc.
1. Inactivating mutations in components of SMAD pathway: SMAD4, a tumor suppressor gene, is deleted or
mutated in pancreatic carcinoma. Mutant protein cannot form transcriptionally active DNA binding complex.
SMAD2 mutation is found in colon, head, neck, lung carcinoma. In breast cancer, inactivating mutation of SMAD4 is
rare and SMAD2 is not found. It is still unknown why inactivating mutations in TGFβR-II and SMAD4, respectively,
are uncommon in breast (and other) cancers but play a substantial role in considerable percentages of gastrointestinal
and pancreatic cancers (Kretzschmar, 2000).

32. TGF-β and its association with cancer
2. Activation of Ras protein:
TGF-β can activate Ras–Mapk signaling (Arteaga, 2006). Also, oncogenic Ras gene interfere with the
phosphorylation of SMAD3 and results in proliferative function of TGF-β. Mutation of Ras protein result in
permanently activated Ras protein which cause SMAD3 phosphorylation and cell proliferation
(Kretzschmar, 2000).
3. Alteration in expression of SMAD inhibitor:
SMAD 6 &7, which are inhibitors of SMAD signaling pathway and inhibit phosphorylation of SMAD2 and
SMAD3, cause tumor cell proliferation in pancreatic carcinoma (Kleff et al., 1999; Kretzschmar, 2000).
4. Stimulation of epithelial mesenchymal transmission (EMT) (metastasis): Increased expression of
SMAD3 & 4 induce EMT (Deryck & Zhang, 2014).

33. How TGF-β induces Epithelial Mesenchymal Transition
1. TGF-β acts as a common and potent inducer of EMT via Smad-dependent and independent activation of the
expression of EMT transcription factors Snail, Slug, ZEB1 and 2, and Twist . The Smad3/4 complex directly binds to
the regulatory portion of the promoter of Snail, inducing its transcription. Subsequently a Smad3/4/Snail complex is
formed that binds to the regulatory promoter sequences of genes encoding for E-cadherin and occludin, leading to
repression of their expression.
2. Smad signaling also increases the expression of ZEB transcription factors, which repress miR-200 family
expression, further increasing ZEB protein levels and EMT.
3. Moreover, TGF-β can activate EMT transcription factor expression via alternative splicing.

34. 4. EMT is also controlled by a group of microRNAs that define changes in cytoskeleton reorganization and
epithelial polarity, and it is directly activated in response to TGF-β via the Smad/RhoA pathway.
5. TGF-β also induces EMT through ubiquitylation and sumoylation. Smad3/4 complex regulates the expression of
HDM2, increasing the ubiquitylation and degradation of p53, inducing EMT progression. TGF-β signaling
downregulates the expression of the SUMO E3 ligase.

35. How TGF-β polymorphism is related to resistance to chemotherapy?
• Inactivating mutations in components of the TGF-β/SMAD signaling pathway
• Reduced expression of TGF-β/SMAD signaling components
• Inhibition of the TGF-β/SMAD pathway by Ras/MAP-kinase signaling
• Altered expression of TGF-β/SMAD inhibitory molecules
• Interference with TGF-β/SMAD responses downstream of SMAD proteins

36. ● Anticancer drugs can become resistant to apoptosis due to anti-apoptotic state induced by TGFβ. Such as
oxaliplatin.
● TGFβ can also prevent chemotherapy induced apoptosis by inhibiting tumor suppressor gene p53 directly and
indirectly.
● In case of paclitaxel, it may become resistant due to increased expression of TGFβ signaling component.

37. Cross talk with other pathways
 TGF-β and MAPK Pathways:
One of the stimulating factors of MAPK pathway is, Ras protein can antagonize TGF beta induced apoptosis and
cell cycle arrest, resulting in cancer cell proliferation. The synergistic relation between TGF-β and RAS/ MAPK
pathways lead to cytokines and growth factors and thereby epithelial to mesenchymal transition.
 TGF-β and PI3/AKT pathway:
PI3/AKT reduce the TGF-β induced cell cycle arrest and apoptosis. SMAD3 is inhibited by PI3/AKT (Guo et al.,
2009).

38. Deregulation of major components of mTOR pathway leading to chemoresistance
Molecular events Resistant to Tumor/cell types
Activation of PI3K pathway,
either via loss of the tumor
suppressor PTEN or through
amplification of the PI3K
encoding PIK3CA gene
Trastuzumab Breast cancer
PI3K overexpression and
PTEN reduction
Cisplatin Ovarian cancer
PI3K regulates MDR1
expression
Vincristine Leukemia, prostate cancer
Activation of PI3K and AKT Endocrine
therapy
Breast cancer

39. Deregulation of major components of mTOR pathway leading to chemoresistance (Cont.)
Molecular events Resistance
to
Tumor/cell types
Activation of
PI3K and AKT
Imatinib Gastrointestinal stromal tumor
Constitutive AKT
activation
Tamoxifen Breast cancer
Constitutive AKT
activation
TRAIL Leukemia, prostate cancer
AKT inhibits p53
phosphorylation
Cisplatin Ovarian cancer

40. Molecular events Resistance to Tumor/cell types
Overexpression of
mTOR and p70S6K1
TRAIL Glioblastoma
Defect in the
regulation of 4EBP1
and 4EBP2
Retinoic acid NB4 cell
mTOR activation Vincristine FL5.12 cells
Raf activation Doxorubicin
and paclitaxel
Breast cancer
MEK/ERK regulates
MDR1 expression
Doxorubicin
and paclitaxel
Colorectal cancer
Deregulation of major components of mTOR pathway leading to chemoresistance (Cont.)

41. Classification of mTOR inhibitors

42. The Wnt/β-catenin pathway
The name Wnt is a portmanteau of “int” and “Wg” and stands for "Wingless-related integration site". Wnts are
secreted factors that regulate cell growth, motility, and differentiation during embryonic development. β-Catenin
is an extremely important effector in the Wnt signaling pathway.

43. When Wnt proteins are present, the so-
called Wnt-on state, the Wnt ligands
bind to FZD and LRP5/6 on the cell
membrane. The binding of Wnt to FZD
exposes an intracellular binding site for
DVL in FZD. A destruction complex
exists in the cytoplasm, consisting
mainly of AXIN, the tumor suppressor
gene APC, GSK3β and CK1α. In Wnt-
on state, the DVL protein inhibits the
destruction complex. Subsequently,
GSK3β and CK1α can not
phosphorylate β-catenin. This results in
functional β-catenin accumulating in
the cytoplasm and entering the nucleus
with the assistance of relevant
molecules. Thereafter, β-catenin
regulates the transcription of
transcriptional regulators such as
TCF/LEF and genes that are ultimately
targeted by Wnt.
The Wnt-on State

44. Phosphorylation of β-catenin exposes a binding site for the E3 ubiquitin ligase β-TrCP, and β-catenin is thus
ubiquitinated and degraded, as such, it is unable to enter the nucleus to initiate downstream gene transcription.
In the absence of Wnt proteins, the so-called
Wnt-off state, AXIN acts as a scaffolding
protein that binds β-catenin, and AXIN also
binds GSK3β, CK1α, and APC. CK1α and
GSK3β can sequentially phosphorylate β-
catenin, and APC then ensures that
phosphorylated β-catenin is not
dephosphorylated by PP2A and later
phosphorylation of β-catenin.
The Wnt-off State

45. Crosstalk involving the Wnt/β-catenin pathway
Evidence of crosstalk:
The Notch and Wnt signal transduction pathways can regulate each other and hence affect their transcriptional
output.
Notch and Wnt signaling pathways act together during wing development in Drosophila.
In mouse, the Wnt pathway regulate the expression of the Notch ligand: Delta-like ligand1 (DLL1).
• The Notch target gene Hes1 is also regulated by β-catenin-mediated Wnt signaling.
• There is direct interaction between β-catenin and Notch-1.
• Studies have shown Notch/RBP-J/β-catenin interaction at several target genes and synergism of them in
angiogenesis.

46. Influence of Notch on β-catenin
Notch tethers β-catenin Negative regulation
• Notch upregulates the mRNA levels of the canonical Wnt-transcription
factor TCF1.
• Frizzled receptor is activated by Notch signaling in dendritic cell.
• The Notch target gene Nrarp acts as both positive & negative regulator on
Wnt.

47. Influence of β-catenin on NOTCH
Wnt/Ca2+ pathway
mediated
activation of
Frizzled
Release of Ca2+ Activation of
CaMKII
Activation of the β-
catenin-pathways
Phosphorylation of
the RBP-J-
interacting co-
repressor SMRT
Increased
promoter activity
of a Notch receptor