1. Cell Cycle and Apoptosis
October 12, 2017

2. Classes of Bcl2 Proteins
Bcl2 proteins –regulate apoptosis through controlling the release of cytochrome c

3. Pro-Apoptotic BH123

4. Regulation of Intrinsic Pathway
BH3-only proteins activate apoptosis through direct binding with anti-apoptotic proteins
-p53 activates BH3-only proteins (Puma and Noxa)
-Bid – extrinsic and intrinsic pathways

5. BNIP3 Family -BH3-only protein, pro-apoptotic Family memebers:
BNIP3 (Bcl-2/EiB-19K interacting protein 3)
BNIP3L, B5, BNIP3α, NIX, BNIP3h

6. Regulation of BNIP3 family
-Up regulated in hypoxic conditions, 2-fold
-HIF-1α activates BNIP3
-PLAGL2 can also activate BNIP3

7. Regulation of BNIP3 family
How does Apoptosis lead to Cancer?

8. Regulation of BNIP3 family
How does Apoptosis lead to Cancer?
Hypoxic stress in the tumor microenvironment contributes to the evolution of malignant tumor cells with increased invasive and angiogenic potentials
Correlation between tumor hypoxia and increased metastatic efficiency and resistance to radiation and chemotherapy
Conflicting signals between:
Death factors Vs. Survival factors
BNIP3 VEGF, FGF, IGF

9. Regulation of BNIP3 family
Inactivation or Down regulation of BNIP3

10. Regulation of BNIP3 family
Pancreatic Cancers
-Hypermethylation of BNIPs promoter
-Other hypoxia induced genes were up regulated
Colorectal Cancers
-histone deacetylation

12. Extrinsic Pathway
DISC – death-inducing signaling complex
Inhibitors such as decoy receptors and intracellular blocking proteins

13. TRAIL Extrinsic Apoptotic Pathway

14. TRAIL Tumor necrosis Factor-related apoptosis-inducing ligand (TRAIL)
-Induction of Extrinsic
Apoptotic pathway
-Many cancers are sensitive
to TRAIL-induced apoptosis
-Some are not:
Pancreatic cancer, melanoma,
and neuroblastoma

15. TRAIL Receptors 5 Receptors
-DR4 and DR5, have the Death Domain to trigger apoptosis
-DcR1 and DcR2, Decoy Receptors, cannot trigger apoptosis
-OPG, secreted and detected in circulation
DR and DcR are both widely expressed in many human cells

16. Single Nucleotide Polymorphisms SNPs
-a DNA nucleotide variation occurring in the genome between two members of the same species

17. Occurrance in Humans -1 in 1000 bases, 0.1%
-3 to 10 million SNPs in the
human genome

18. Importance of SNPs

19. Cancer Susceptibility

20. Polymorphisms in DR4 Receptors
Ovarian and Bladder Cancer Cell lines
-A to G change in DR4
-resulting in K441R mutation, Lysine to Arginine
-Less effective in cell killing

21. Polymorphisms in DR4 Receptors
Lung, head and neck, and gastric adenocarinoma
-C to G change in DR4
-resulting in T209R, Threonine to Arginine
-G to A change in DR4
-resulting in R141H, Arginine to Histidine
-Abnormal death receptor trimerization
Homozygous for both T209R and R141H
-13% Normal individuals
-35% non-small cell lung cancer
-47% primary head and neck cacner
-44% gastric adenocarcinomas

22. Mutations in DR4 Receptors
Breast Cancer specimens
-34 specimens which metastasized
-3 mutations in DR4
-single base changes in the death domain
-Also showed an increase in LOH
-23 specimens had not metastasized
-0 mutations in DR4

23. Mutations in DR5 Receptors
Primary head and neck cancer
-40 specimens
-2 mutations in DR5
-A 2 base insertion in death domain
-Truncated protein
Non-small cell lunch cancer
-104 specimens
-11 mutations in the death domain of DR5
-7 of the 11 showed LOH

24. Mutations in DR5 Receptors
Breast Cancer specimens
-34 specimens which metastasized
-4 mutations in DR4
-2 were in the death domain, 2 adjacent
-Also showed an increase in LOH
-23 specimens had not metastasized
-0 mutations in DR4
Placement of these mutations in other cells resulted in suppression of apoptosis

25. TRAIL Resistance: Caspase-8
-Down regulation of Caspase-8 in TRAIL Resistant Cancers
-Gene deletion (rare)
-Promoter methylation
- Treatment of cells with demethylating agent restored apoptosis
Caspase-8 importance
-Caspase-8 Inhibitor, Z-IETD-FMK
halted apoptosis
-Caspace-10 was not a
functional substitute X

26. TRAIL Resistance: Bcl-2 family
-Up regulation of Bcl-2 or Bcl-XL
-Pancreatic adenocarcinoma
-Bcl-XL was up regulated in 3 cell lines
Neuroblastoma, glioblastoma,
and breast cancer
-Bcl-2 was up regulated in
cell lines X

27. TRAIL Resistance: Bax and Bak
-Bax and Bak are pro-apoptotic genes
-They have redundant functions as seen in Bax -/- or Bak -/- mice
-To result in TRAIL-induced apoptosis,
the mice must be both Bax -/- Bak -/-
-They would need to be down
regulated X

28. TRAIL Resistance: Bax and Bak
-Bax and Bak are pro-apoptotic genes
-They have redundant functions as seen in Bax -/- or Bak -/- mice
-To result in TRAIL-induced apoptosis,
the mice must be both Bax -/- Bak -/-
-They would need to be down
regulated X

29. TRAIL Resistance: IAPs and anti-IAPs
-IAPs have domains called baculovirus inhibitory repeat (BIR) thought to interact with different caspases
-Trail Resistance by:
-Up regulation of IAPs
-Down regulation of anti-IAPs X X

30. TRAIL Resistance: IAPs

31. TRAIL Resistance: NF-κB
Controversial

32. TRAIL-Induced Apoptosis