NSAIDs therapy for colorectal cancer: mechanisms and efficacy PHM142 Fall 2016 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson NSAIDs therapy for colorectal cancer: mechanisms and efficacy PHM142 Sign up date: Oct 17, 2017 Ji Hyun (Sera) Lee Cathy Hoang Janet Chan Rui Si (Louise) Pi

What are NSAIDs? → Non-steroidal anti-inflammatory drug → Commonly used for fever, inflammation, pain → Examples: Acetylsalicylic Acid (Aspirin), Ibuprofen (Advil), Naproxen (Aleve) → Some available as OTC

NSAIDs’ Molecular Targets - COX enzyme → Cyclooxygenase enzyme (COX) responsible for the production of prostaglandins via arachidonic acid → Prostaglandins will activate the inflammatory response, resulting in fever/pain → Also play a role in constricting blood vessels, aggregating platelets (blood clotting) → NSAIDs bind to active sites on COX, thereby preventing the formation of prostaglandins, and reducing inflammation, fever, and pain → Aspirin irreversibly acetylates COX binding site, resulting in blood thinning, and less clotting

→ Prostaglandin synthesis pathway → Production of various prostaglandins and thromboxanes → Thromboxane aids in blood clotting and vasoconstriction of vessels

Colorectal Cancer and Wnt-Signaling pathway → 2nd most commonly diagnosed cancer in Canada → On average, 26 Canadians die from colorectal cancer daily → Malignant tumour starts in colon/rectum → NSAIDs can aid in reducing cell proliferation and thus preventing cancer under normal physiology, wnt signaling protein would bind to the g-protein, no complex is made of APC-axin-GSK, resulting in beta-catenin not degraded so it can travel into the nucleus and turn on transcripton for cell proliferation. when theres no wnt, beta-catenin is targeted for degradation by APC-axin-GSK complex→ B catenin is phosphorylated + degraded = no cell proliferation in cancer pts, mutation on APC gene --> APC loss of function = no complete complex + no phosphorylation of B catenin --> so beta-catenin will never get degraded --> uncontrolled cell proliferation

Inhibition of COX2: immune response suppression and subsequent inhibition of Wnt-signalling pathway PGE2 promotes malignant cell growth by mediating immune response Mucosal injury on the colorectal epithelium of CRC patient Immune cells being recruited to site of injury Cytokines (IL-alpha, IL-beta, interferon-gamma) released Activation of COX2 and PGE2 synthesis PGE2 binds to its receptor EP2 (G-protein coupled receptor) Complexing of axin, APC and G-protein Accumulation of beta-catenin in the cytosol and nucleus Beta-catenin activates the transcription of its target genes, leading to uncontrolled cancerous cell proliferation EP2 As we mentioned, NSAIDs could inactivate COX-2, thereby inhibiting prostaglandin E2 synthesis (PGE2) As sera mentioned, in colorectal cancer pts, the Wnt-signalling pathway is unregulated, leading to uncontrolled proliferation of cancerous cells It was found that PGE2 plays an important role in promoting cancerous cell growth by mediating an immune response and by strengthening the Wnt-signalling pathway (left) when there is mucosal injury in the colorectal epithelium, immune cells(such as neutrophils, would be recruited to the site of injury These immune cells then release cytokines (such as interleukin 1 IL‑1α and β, interferon‑γ and tumour necrosis factor (TNF)) In response to those cytokines, COX2 will then be activated and produce PGE2 PGE2 would bind to its receptor EP2 (a G-protein coupled receptor), the binding would lead to the complexing of Axin, APC and the g protein (G alpha S) This will result in the buildup of beta-catenin in the cytosol and nucleus Once in the nucleus → uncontrolled cellular proliferation, and angiogenesis

Inhibition of COX2: immune response suppression and subsequent inhibition of Wnt-signalling pathway Under NSAID therapy COX2 inhibition → PGE2 synthesis ↓ Unbound EP2 liberates APC and axin APC-axin-GSK complex phosphorylates beta-catenin, following by its degradation Without beta-catenin, the tumorigenesis transcription machinery is turned off EP2 In the presence of NSAID, COX2 would be inhibited thereby reducing the production of PGE2 When PGE2 is not bound to the G-protein coupled receptor, Axin and APC are liberated, and they can form a complex with another protein GSK-3beta This new complex would phosphorylate beta-catenin, followed by the degradation of beta-catenin w/o beta-catenin, the tumorigenesis transcription machinery will be turned off

Inhibition of COX1: Platelet inhibition and PEG2 synthesis suppression Platelets Host immune cells Express COX1 only Not nucleated; cannot resynthesize COX1 when inhibited Convert AA to thromboxane TXA2 by COX1 TXA2 Stimulates activation of new platelets as well as increases platelet aggregation Results in more platelets at site of injury and increases PGE2 level With NSAID therapy COX1 inhibition → TXA2 synthesis ↓ → less platelet aggregation at site of injury → COX2 activation and PGE2 synthesis ↓ → Wnt-signalling pathway inactivated → tumorigenesis suppression NSAID COX1 Platelet inhibition Unlike epithelial cells which express both COX-1 and 2 Platelet only expresses COX1 And because it’s not nucleated, it cannot resynthesize COX1 when it is inhibited COX1 converts AA to thromboxane TXA2 (TXA2 stimulates activation of new platelets as well as increases platelet aggregation) - like a positive feedback mechanism Platelets (one of the host immune cells that could activate COX-2 and subsequent PGE2 production) When NSAID is present, inhibits COX-1 → reduce synthesis of TXA2 → less platelet aggregation + no activation of new platelets → no COX-2 activation and reduced PGE2 production → no Wnt-signalling pathway not activated → no cell proliferation

Efficacy of NSAIDs on Colorectal cancer Study by Sheng et al., Growth of human colon cancer cell (HCA-7) that express COX2 in nude mice +/- treatment with selective COX2 inhibitor (SC-58125) COX2 enzyme converts arachidonic acid to prostaglandin: prostaglandin production was significant in HCA-7, but was inhibited by COX2 inhibitor treatment With the addition of COX2 inhibitor, it inhibited the size and number of colonies derived from COX2 expressing HCA-7 Treatment of COX2 inhibitor inhibited tumor development by 90% in HCA-7 This particular study showed how effective NSAIDs is on the treatment of colorectal cancer. The experiment was conducted using growth of human colon cancer cells that constitutively express COX-2 enzyme in nude mice with and without treatment of a highly selective COX-2 inhibitor. Results showed that prostaglandin production was significant in colon cancer cells and this was inhibited by COX-2 inhibitor treatment. Next, non-transformed intestinal epithelial cells are unable to survive when plated in extracellular matrix component. On the other hand, colon cancer cells are able to form discrete colonies when plated. With the addition of COX-2 inhibitor, it dramatically inhibited the size and number of colonies derived from COX-2 expressing colon cancer cells. Finally, treatment of COX-2 inhibitor inhibited tumor development by 90% for human colon cancer cell in the mice. Therefore, COX-2 inhibitor can inhibit colon cancer cell growth in culture as well as tumor implanted in nude mice.

Efficacy of NSAIDs on Colorectal cancer Epidemiologic studies show a 40-50% reduction in mortality from colorectal cancer in individuals taking NSAIDs, compared to those not using it Patients with familial adenomatous polyposis (FAP) who take sulindac have a significant reduction in adenoma size and number Epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in individuals taking NSAIDs, such as aspirin, compared to those not taking these agents And patients with familial adenomatous polyposis (FAP) who take sulindac, also a NSAIDs medication, have a significant reduction in adenoma size and number.

Adverse Effects of NSAIDS → Varies depending on the inhibited COX enzyme(s) → GI Mucosal Effects (COX1 inhibition) → Renal Effects (COX1&2 inhibition) → Cardiovascular Effects (COX1&2 inhibition) There are a lot of adverse effects or side effects that come with using NSAIDs Three large categories of adverse effects are concerning the GI mucosa, kidney and cardiovascular system The effect depends on the COX enzyme that is inhibited

COX1 inhibition = GI bleeding and peptic ulcers GI Mucosal Effects → COX1 is involved with gastric protection → COX1 produces PGE2 → Increases mucus secretion → Increases bicarbonate secretion → Increases mucosal blood flow → Inhibition of COX1/PGE2 production = mucosal and epithelial damage COX1 inhibition = GI bleeding and peptic ulcers COX1 is involved with gastric protection by producing prostagladin E2 Prostaglandin E2 is involved with increasing mucus secretion, bicarbonate secretion and mucosal blood flow By using NSAIDs, COX1 will be inhibited, which subsequently inhibits prostaglandin E2 This leads to muscoal and epithelial damage, and ultimately GI bleeding and peptic ulcers This resulted in the development of selective inhibitors of COX2, such as celecoxib, which has been shown to be effective for colorectal cancer

COX1&2 inhibition = Hypertension and hemodynamic acute kidney injury Renal Effects → COX1&2 involved in PGE2 and PGI2 production → PGE2 and PGI2 have effects on kidneys → Increased sodium and water excretion → Increased afferent arteriolar vasodilation → Inhibition of COX1&2 = sodium and water retention and decreased GFR COX1&2 inhibition = Hypertension and hemodynamic acute kidney injury In terms of renal effects, both COX enzymes are involved COX1 and 2 are responsible for producing prostaglandin E2 and I2 These prostaglandins increase sodium and water excretion, as well as vasodilation of the afferent arteriole Inhibition of COX1 and 2 therefore lead to sodium and water retention and a decrease in glomerular filtration rate Thus, the result of using NSAIDs is hypertension and hemodynamic acute kidney injury since renal perfusion is not maintained

Cardiovascular Effects → COX1 involved in TXA2 production and COX2 involved in PGI2 production → TXA2 effects → Increased platelet aggregation → Increased vasoconstriction → PGI2 effects → Decreased platelet aggregation → Increased vasodilation Inhibition of COX2 = disrupted balance = cardiovascular risks Finally, both COX enzymes are also involved cardiovascular effects COX1 produces thromboxane A2 and COX2 produces prostaglandin I2 Thromboxane increases platelet aggregation and vasoconstriction, whereas prostaglandin I2 decreases platelet aggregation and increases vasodilation By inhibiting COX2 with an NSAID, the balance between thromboxane A2 and prostaglandin I2 is disrupted

Cardiovascular Effects Thromboxane A2 accumulates Clumping will occur and blood vessels with be constricted This can lead to CV risks, such as myocardial infarctions People were focused on preventing GI effects, so they created selective COX2 inhibitors, but didn't look into cardiovascular effects But of course it is important to take this cardiovascular effect into account

Summary NSAIDs bind to COX resulting in reduced inflammation, pain, fever, thinned blood, and blood clotting In colorectal cancer patients, APC is absent, thereby preventing the degradation of beta-catenin which results in cell proliferation PGE2 synthesis induced by inflammation would activate Wnt-signaling pathway, resulting in tumorigenesis Inhibition of COX2 by NSAIDs leads to reduced PGE2 synthesis, beta-catenin degradation and reduced tumorigenesis Inhibition of COX1 by NSAIDs reduces TXA2 synthesis and further platelets activation, COX2 is not activated to produce PGE2, thereby inhibiting Wnt-signalling pathway and malignant cell proliferation COX-2 inhibitor decreases prostaglandin production in colon cancer cells, and inhibits tumor development There is a 40-50% reduction in mortality from colorectal cancer in individuals taking NSAIDs Inhibition of COX by enzymes has GI mucosal, renal and cardiovascular effects Inhibition of COX1 can lead to GI bleeding and peptic ulcers Inhibition of COX1 and 2 can result in hypertension and hemodynamic acute kidney injury Inhibition of COX1 and 2 can cause myocardial infarctions

References Sheng, H., Shao, J., Kirkland, S. C., Isakson, P., Coffey, R. J., Morrow, J., … DuBois, R. N. (1997). Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2. J Clin Invest, 99(9), 2254–9. https://doi.org/10.1172/JCI119400 DuBois, R. N., & Smalley, W. E. (1996). Cyclooxygenase, NSAIDs, and colorectal cancer. Journal of Gastroenterology. https://doi.org/10.1007/BF02358623 Canadian Cancer Society's Advisory Committee on Cancer Statistics. (2017). Canadian Cancer Statistics 2017. Toronto, ON: Canadian Cancer Society Ricciotti, E., & Fitzgerald, G. A. (2011). Prostaglandins and inflammation. Arteriosclerosis, Thrombosis, and Vascular Biology, 31(5), 986–1000. https://doi.org/10.1161/ATVBAHA.110.207449 Abdulghafar, A. Mechanism of action of Aspirin as antiplatelet and its different doses. Retrieved from: http://www.fadic.net/en/content/mechanism-action-aspirin-antiplatelet-and-its-different-doses NSAIDS (nonsteroidal anti-inflammatory drugs). Retrieved from: https://www.rheumatology.org/I-Am-A/Patient-Caregiver/Treatments/NSAIDs Major Side Effects of NSAIDs & COX-2 Selective Inhibitors (2017, March 10). Retrieved from: http://tmedweb.tulane.edu/pharmwiki/doku.php/nsaid_side_effects Clevers, H. (2006). Colon Cancer — Understanding How NSAIDs Work. New England Journal of Medicine, 354(7), 761–763. https://doi.org/doi:10.1056/NEJMcibr055457 Drew, D. A., Cao, Y., & Chan, A. T. (2016). Aspirin and colorectal cancer: the promise of precision chemoprevention. Nature Reviews Cancer, 16(3), 173–186. https://doi.org/10.1038/nrc.2016.4 Weinberg, J., Fermor, B., & Guilak, F. (2007). Nitric oxide synthase and cyclooxygenase interactions in cartilage and meniscus: relationships to joint physiology, arthritis, and tissue repair. Sub-Cellular Biochemistry, 42, 31–62. https://doi.org/17612045