Tumor Signaling - Detailed Explanation

SLIDE 3 - Types of Mutations

Point Mutations

Chromosomal Mutations

• Deletion: Loss of a chromosomal region - if it contains a tumor suppressor gene (e.g., RB1 on chromosome 13, TP53 on chromosome 17p), the cell loses that growth brake. This follows Knudson's "two-hit hypothesis" - both alleles must be lost for tumor suppressor function to be abolished.
• Duplication: Amplification of a proto-oncogene - e.g., MYC amplification in Burkitt's lymphoma, HER2 amplification in breast cancer, drives overproduction of growth-promoting proteins.
• Inversion: A segment flips orientation - can place a normally regulated gene under a different (stronger) promoter, changing its expression.
• Translocation: A fragment moves between chromosomes - the classic example is the Philadelphia chromosome (t(9;22)), which fuses BCR to ABL1, creating a constitutively active tyrosine kinase that drives chronic myelogenous leukemia (CML). Another example: t(8;14) in Burkitt's lymphoma places MYC under the control of the immunoglobulin heavy chain promoter.

SLIDE 4 - Epigenetic Changes

DNA Methylation

• Hypermethylation of a gene's promoter physically blocks transcription factor binding and recruits methyl-CpG-binding proteins that compact chromatin - effectively silencing the gene. In cancer, this commonly silences tumor suppressor genes:
  • CDKN2A (p16) - loss allows unchecked CDK4/6 activity and Rb phosphorylation
  • MLH1 - a mismatch repair gene; silencing leads to microsatellite instability (seen in Lynch syndrome-type colorectal cancers)
  • BRCA1 - silencing mimics a BRCA1 mutation in sporadic ovarian/breast cancers
• Hypomethylation globally across the genome is also seen in cancer. It destabilizes chromosomes and can reactivate oncogenes and transposable elements that are normally silenced.

Histone Modification

• Acetylation (by HATs - histone acetyltransferases) loosens chromatin, activating transcription
• Deacetylation (by HDACs - histone deacetylases) compacts chromatin, silencing genes
• Methylation of H3K27 (by EZH2, a polycomb protein) is a repressive mark - overexpression of EZH2 in cancer silences tumor suppressor gene clusters
• The PPT's point about "overpacking suppressing tumor suppressor genes" refers to this: excessive deacetylation or repressive methylation condenses chromatin around tumor suppressor loci, preventing their transcription even when the DNA sequence is intact.

Chromatin Remodeling

SLIDE 5 - Oncoviruses

SLIDE 8 - Cell Proliferation (Key Targets of Mutation)

MSKs (Mitogen and Stress-Activated Kinases 1/2)

• Downstream of ERK1/2 and p38 MAPK pathways
• Phosphorylate histone H3 at Ser10, promoting chromatin decondensation and transcriptional activation of immediate early genes (c-Fos, c-Jun)
• In tumors: overactivation of MSK1/2 amplifies transcription of proliferation genes; phosphorylated H3 is used as a marker of mitosis and proliferative activity

RSKs (Ribosomal S6 Kinases 1/2)

• Downstream of ERK pathway
• Phosphorylate the 40S ribosomal protein S6, stimulating protein synthesis capacity
• Phosphorylate and inactivate BAD (pro-apoptotic protein), promoting survival
• Activate CREB transcription factor, driving expression of cyclin D1 and anti-apoptotic genes
• In cancer: RSK2 is overexpressed in head/neck and prostate cancers; drives resistance to apoptosis

MYC

• A master transcription factor (bHLH-leucine zipper family) that heterodimerizes with MAX
• Activates transcription of hundreds of target genes promoting cell cycle entry (cyclin D, CDK4), ribosome biogenesis (RNA Pol I targets), and metabolism
• Also represses CDK inhibitors (p21, p27) and pro-differentiation genes
• Amplified in up to 30% of all human cancers; in Burkitt's lymphoma, translocation puts MYC under Ig promoter control

GSK3 (Glycogen Synthase Kinase-3) → β-catenin

• GSK3β is normally active and phosphorylates β-catenin, tagging it for ubiquitin-mediated proteasomal degradation
• The WNT signaling pathway inhibits GSK3β - this stabilizes β-catenin, which then translocates to the nucleus and forms a complex with TCF/LEF transcription factors, driving expression of MYC, cyclin D1, and other oncogenes
• In cancer: mutations in CTNNB1 (the β-catenin gene) at GSK3β phosphorylation sites prevent its degradation - this constitutively active β-catenin drives colorectal cancer, hepatocellular carcinoma, and medulloblastoma
• AKT phosphorylates and inhibits GSK3β, so hyperactivation of the PI3K-AKT pathway also stabilizes β-catenin - this is why PI3K/AKT mutations tie into Wnt/MYC-driven proliferation

SLIDE 10 - Cell Survival (Survival Support)

Overexpression of BCL-2

• BCL-2 is an anti-apoptotic protein on the outer mitochondrial membrane that blocks the intrinsic (mitochondrial) apoptosis pathway
• It sequesters pro-apoptotic proteins BAX and BAK, preventing mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release
• Without cytochrome c release, the apoptosome (Apaf-1/cytochrome c complex) cannot form, so caspase-9 and downstream executioner caspases (3, 7) are not activated
• BCL-2 is overexpressed via the t(14;18) translocation in follicular lymphoma; also overexpressed in CLL, multiple myeloma, and many solid tumors
• Venetoclax is a BH3 mimetic drug that competitively inhibits BCL-2, restoring apoptosis in BCL-2-dependent tumors

Loss of p53

• p53 is the "guardian of the genome" - normally it detects DNA damage, oncogene activation, or hypoxia and triggers either cell cycle arrest (via p21/CDKN1A) or apoptosis (via PUMA, NOXA - pro-apoptotic BH3-only proteins)
• TP53 mutations occur in >50% of all human cancers - most commonly missense mutations in the DNA-binding domain that not only abolish p53 function but can have dominant-negative or gain-of-function effects
• HPV E6 protein targets p53 for ubiquitin-proteasome degradation; MDM2 amplification (seen in sarcomas) keeps p53 constitutively degraded
• Loss of p53 also impairs induction of ferroptosis (iron-dependent oxidative cell death), another tumor-suppressive mechanism

Hyperactivation of RAS-ERK and AKT

• RAS-ERK: Activated RAS (via oncogenic KRAS, NRAS, or HRAS mutations) drives RAF-MEK-ERK signaling. ERK phosphorylates and inhibits BIM (a pro-apoptotic BH3-only protein), and activates MCL-1 (anti-apoptotic BCL-2 family member). Net effect: apoptosis is blocked.
• AKT (PKB): Activated by PI3K (downstream of growth factor receptors or PTEN loss), AKT phosphorylates BAD (inactivating it), inhibits FOXO transcription factors (which otherwise drive expression of death genes), activates MDM2 (which degrades p53), and phosphorylates caspase-9 to inhibit it directly.

Phosphorylation of FOXO3A

• FOXO3A is a Forkhead transcription factor that, when active (dephosphorylated), transcribes pro-apoptotic genes (BIM, FasL, TRAIL), cell cycle inhibitors (p27), and genes promoting autophagy
• AKT phosphorylates FOXO3A at three key residues (Thr32, Ser253, Ser315), causing it to bind 14-3-3 scaffold proteins and be exported from the nucleus and degraded in the cytoplasm
• This is one of the key mechanisms by which PI3K-AKT pathway activation promotes cancer cell survival - by neutralizing a transcriptional "death program"
• FOXO3A is also inhibited by IKK (in the NF-kB pathway) and by ERK

SLIDE 11 - Metabolic Changes (Warburg Effect and Beyond)

Increased Glucose Uptake and Glycolysis (Warburg Effect)

• Normal cells use oxidative phosphorylation (OXPHOS) when oxygen is available (30-38 ATP/glucose)
• Cancer cells preferentially use aerobic glycolysis even with normal oxygen - producing only 2 ATP/glucose but generating lactate and, critically, metabolic intermediates (glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate) for biosynthesis
• Driven by: MYC (upregulates LDHA, PKM2, and glucose transporters), HIF-1α (even in normoxia via VHL loss or PI3K activation), and oncogenic RAS
• Lactate export acidifies the tumor microenvironment, impairing immune cell function and promoting invasion

Increased Serine/Glycine Synthesis

• The serine synthesis pathway branches off glycolysis at 3-phosphoglycerate
• Serine feeds into: folate cycle (one-carbon metabolism for nucleotide synthesis), glutathione synthesis (antioxidant defense), sphingolipid metabolism, and protein synthesis
• PHGDH (phosphoglycerate dehydrogenase) is amplified/overexpressed in ~6% of breast cancers and ~10% of melanomas
• Glycine is used to synthesize purines and porphyrins; excess glycine (with serine) also feeds into the mitochondrial serine catabolism pathway to generate NADH and formate

Pyrimidine Synthesis Leading to S-Adenosylmethionine (SAM)

• SAM is the universal methyl donor in the cell - used for DNA methylation, histone methylation, and phosphatidylcholine synthesis
• Pyrimidine/nucleotide synthesis is upregulated via MYC (which transcriptionally activates CAD, the trifunctional enzyme for de novo pyrimidine synthesis) and mTORC1 (which activates CAD via S6K1)
• High SAM production supports the epigenetic changes (methylation patterns) discussed in Slide 4

Overexpression of GLUT4 via SLC2A4

• GLUT4 is the insulin-regulated glucose transporter (normally in adipocytes and muscle)
• Overexpression in cancer cells increases basal glucose uptake independent of insulin
• Driven by MYC overexpression (directly transactivates SLC2A4) and oncogenic PI3K-AKT signaling

IDH1/2 Mutations - 2-Hydroxyglutarate (2-HG)

• Normal IDH1 (cytoplasmic) and IDH2 (mitochondrial) catalyze isocitrate → α-ketoglutarate (αKG) + CO2 + NADPH
• Gain-of-function mutations (R132H in IDH1; R140Q or R172K in IDH2) cause the enzymes to instead reduce αKG to the oncometabolite 2-hydroxyglutarate (2-HG)
• 2-HG competitively inhibits αKG-dependent dioxygenases:
  • TET enzymes (DNA demethylases) → hypermethylation phenotype (CpG island methylator phenotype, CIMP)
  • KDM histone demethylases → histone hypermethylation
  • PHDs (HIF prolyl hydroxylases) → HIF stabilization → pseudo-hypoxic state
• This creates a block in differentiation (cells are "frozen" in a stem-like state), which is the basis of IDH-mutant AML, glioma (grade 2/3), and cholangiocarcinoma
• IDH inhibitors (enasidenib for IDH2, ivosidenib for IDH1) are approved treatments that reduce 2-HG and promote leukemic differentiation

SLIDE 13 - Cell Polarity and Migration Effectors

Rho Family GTPases (RhoA, Rac1, Cdc42)

• Act as molecular switches: active when GTP-bound, inactive when GDP-bound
• RhoA activates ROCK kinase → phosphorylates myosin light chain → actomyosin stress fiber contraction → "amoeboid" migration
• Rac1 activates WAVE/ARP2/3 complex → actin branching → lamellipodia formation (broad, flat protrusions at the leading edge)
• Cdc42 activates N-WASP/ARP2/3 → filopodia (thin spike-like protrusions for sensing direction)
• Formins (mDia family): downstream of RhoA, nucleate unbranched actin filaments to form stress fibers and filopodia
• In cancer: RAC1 mutations (P29S) appear in melanoma; RHOA mutations in diffuse gastric cancer; dysregulation is common across all invasive tumors

Integrins and Matrix Adhesion Proteins

• Integrins are heterodimeric (α/β) transmembrane receptors that bind ECM proteins (fibronectin, collagen, laminin)
• They activate focal adhesion kinase (FAK) and Src kinase at focal adhesion complexes
• FAK phosphorylation recruits paxillin, talin, and vinculin to stabilize adhesions and activates PI3K-AKT and RAS-ERK survival/proliferation pathways
• In cancer: altered integrin expression profiles change which ECM components cells can adhere to, enabling migration into new environments; αvβ3 integrin is upregulated on invasive tumor cells and tumor-associated endothelial cells

Extracellular Proteases

• MMPs (Matrix Metalloproteinases): Zinc-dependent endopeptidases that degrade collagen, gelatin, laminin, and other ECM components. MMP-2 and MMP-9 (gelatinases) are particularly important in cancer invasion.
• uPA (urokinase plasminogen activator): Converts plasminogen to plasmin, which activates MMPs and directly degrades fibronectin and laminin
• Proteolytic ECM degradation: (1) creates physical space for invasion, (2) releases sequestered growth factors (VEGF, FGF, TGF-β stored in ECM), and (3) reduces adhesive contacts (reduces E-cadherin-mediated cell-cell adhesion), releasing cells for migration
• This is why the PPT states "reduction of adhesive contacts causes release and spread"

Cell-Cell Adhesion Complexes

• E-cadherin (encoded by CDH1): Forms adherens junctions between epithelial cells; loss is the hallmark of epithelial-to-mesenchymal transition (EMT)
• EMT is a process where epithelial cells lose polarity and cell-cell contacts and acquire mesenchymal, migratory properties - driven by transcription factors Snail, Twist, ZEB1/2 (which repress E-cadherin transcription)
• Loss of E-cadherin also releases β-catenin from the junctional complex, allowing it to signal via the Wnt pathway (as described in Slide 8)
• Claudins and occludins (tight junction proteins) are also downregulated during invasion

Transcription Factors: AP-1 and ETS-2

• AP-1 (composed of FOS/JUN dimers): Activated by RAS-ERK and PKC; transcribes MMP-1, MMP-3, MMP-9, cyclin D1, uPA - directly linking growth factor signaling to the invasion machinery
• ETS-2: A transcription factor activated by RAS-ERK-RSK signaling; upregulates MMP-1, MMP-9, uPA, and VEGF; marks a cancer cell that has activated the entire invasive and angiogenic transcriptional program

SLIDE 14 - Tumor Microenvironment (TME)

Extracellular Matrix (ECM) Disruption

• Normal ECM sequesters growth factors (VEGF, FGF-2, HGF, TGF-β) in bound, inactive forms
• When MMPs degrade the ECM, these factors are released in bioactive form, creating a "growth factor reservoir" that is unleashed during invasion
• ECM stiffness itself (increased by fibrosis and collagen crosslinking by LOX enzymes) activates integrin-FAK signaling and promotes a more malignant phenotype

Angiogenesis

• Tumors cannot grow beyond ~1-2 mm without a blood supply - this triggers the "angiogenic switch" (balance tips from anti-angiogenic to pro-angiogenic signals)
• Key pro-angiogenic factors:
  • VEGF (Vascular Endothelial Growth Factor): The master angiogenic driver; binds VEGFR-2 on endothelial cells → RAS-ERK, PI3K-AKT, PLCγ signaling → EC proliferation, migration, survival, and vascular permeability. Induced by HIF-1α (hypoxia), RAS, MYC, and PDGF
  • PDGF (Platelet-Derived Growth Factor): Recruits pericytes to stabilize new vessels; also directly stimulates tumor cell proliferation
  • FGF (Fibroblast Growth Factor): Released from ECM upon MMP activity; synergizes with VEGF to promote EC survival and tube formation
• Anti-VEGF therapy (bevacizumab, ramucirumab) and VEGFR-TKIs (sunitinib, sorafenib) exploit this pathway therapeutically

Inflammation

• Tumor-associated macrophages (TAMs), neutrophils, and mast cells are recruited by cancer cell-secreted chemokines (e.g., CCL2, CXCL8)
• Once in the TME, TAMs are polarized to an M2-like phenotype (anti-inflammatory, pro-tumorigenic) - they secrete EGF, HGF, VEGF, and IL-10, all of which promote tumor growth and suppress anti-tumor immunity
• Cancer cells also secrete IL-6 and TNF-α, activating NF-kB in both tumor cells and stromal cells, creating a self-reinforcing inflammatory loop that promotes survival, proliferation, and angiogenesis
• Chronic inflammation = chronic DNA damage from ROS/RNS = increased mutation rate

Cancer-Associated Fibroblasts (CAFs)

• Recruited from normal fibroblasts (and bone marrow-derived mesenchymal stem cells) by TGF-β, PDGF, and FGF secreted by tumor cells
• CAFs secrete: hepatocyte growth factor (HGF, activating MET receptor), SDF-1/CXCL12 (recruits endothelial progenitor cells for angiogenesis), IL-6 (activates STAT3 in tumor cells), and produce ECM components (collagen, fibronectin) that stiffen the stroma
• CAFs also produce MMPs, directly remodeling the ECM to facilitate invasion
• CAF-derived TGF-β is a major driver of resistance to chemotherapy and immunotherapy

Summary: How These Pathways Connect

Growth Factor Receptor Activation
        ↓
   RAS activation → RAF-MEK-ERK → MSK1/2, RSK1/2
        ↓                              ↓
  PI3K-AKT-mTOR                 MYC, AP-1, ETS-2
        ↓                              ↓
   FOXO3A inhibited          Proliferation genes, MMPs
   GSK3β inhibited → β-catenin → Wnt target genes
   BAD phosphorylated → BCL-2 family → anti-apoptosis
        ↓
  IDH mutation → 2-HG → TET/KDM inhibition → epigenetic silencing
        ↓
  Warburg metabolism → serine/glycine, pyrimidines for growth
        ↓
  Rho GTPases, integrins, MMPs → ECM invasion
        ↓
  VEGF, PDGF, CAFs → angiogenesis, TME remodeling

Source: Harrison's Principles of Internal Medicine 22E (2025), Chapter 77 - Cancer Cell Biology