Published online on November 20, 21012; Link to the original blog:

http://www.sciclips.com/sciclips/blogArticle.do?id=1024&blog=Cancer%20Biomarker%20Strategy%20to%20Develop%20Companion %20Diagnostics%20for%20Predicting%20Prescription%20Drug%20Induced%20Tumors%20%20Analysis%20using%20pioglitazon%20%28Actos%29%20and%20bladder%20cancer

Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors Analysis using pioglitazone (Actos) and bladder cancer
Cancer biomarkers can be used for developing assays for clinical diagnosis, identifying patients response to a particular drug, optimizing personalized drug treatment regimen (drug dose, drug treatment schedule etc.), monitoring the efficacy of treatment (disease stage, tumor progression, tumor recurrence etc.) and in cancer theranostics (1).With the growing trend towards the advancement of personalized medicine concept, companion diagnostic tools may play a significant role in patient stratification by identifying patients with positive clinical response to an existing or novel treatment method. However, current limitations in identifying life-threatening side effects of therapeutic drugs may have negative impact on developing efficient drug therapy strategies, often difficult to identify short or long term side effects of drugs during clinical trials. Therefore, there is a need for developing predictive methods and assays for identifying secondary disease causing side effects of drugs. We propose disease specific diagnostic biomarkers as an attractive tool for predicting the occurrence of secondary diseases from a specific drug treatment method. In one of our earlier blogs, we have proposed that drug-efficacy/drug response biomarkers might be possibly used for predicting disease causing side effects of therapeutic drugs (2). In this blog, we tried to explore the potential of cancer diagnostic biomarkers for predicting therapeutic drug (non anti-cancer drugs) induced cancer occurrence in patients. We have adopted hypothesis driven intelligent data mining and contextual functional mapping of biomolecules associated with biomarkers and drug targets to identify possible pathways and biomolecules that are associated with prescription drug induced tumor formation. Strategic identification of such biomolecules might lead to the development of companion diagnostic tools that may help in identifying patients at risk of developing cancer following a therapeutic regimen. Moreover, success in this approach will also help in identifying alternative therapies, such as combination therapy, and novel therapeutic drug targets. In order to test our hypothesis, we chose the anti-diabetic drug pioglitazone (Actos), which has been reported to form bladder cancer in patients (3)(4)(5)(6)(7)(8)(9)(10). For identifying biomolecules that might be potentially associated with pioglitazone induced bladder cancer development in diabetic patients, hypothesis driven functional

1 | P a g e Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors h t t p : / / w w w . s c i c l i p s . c o m - Analysis using pioglitazone (Actos) and bladder cancer

integration and identification of biomolecules, incorporating traditional pathway analysis, linked to bladder cancer specific diagnostic biomarkers and drug target (PPARgamma) were adopted (Fig. 1). Several diagnostic biomarkers have been reported for bladder cancer, among these biomarkers we have randomly selected Cox-2(11), metalloproteinases (12)(13) and PGE2 (14) for this theoretical exploratory analysis. A. Potential companion diagnostic biomarkers for predicting tumor inducing properties of therapeutic drugs (e.g. pioglitazone) 1. Prostaglandin2 (PGE2) and PGE2 induced bladder cancer stem cells Pioglitazone (Actos) is a thiazolidinedione (TZD) anti-diabetic drug that selectively activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma). Like other TZDs, pioglitazone induces insulin-sensitive genes (transcriptional regulation) associated with control of glucose and lipid metabolism in muscle, adipose tissue, and the liver through the activation of PPAR gamma (15). Studies have shown that expression of PPAR gamma represses activator protein-1 (AP1) and nuclear factor kappa B (NF-kappaB) transcriptional activity. Down-regulation of PPAR gamma induces the expression of COX-2 (16)(17), the PGE2-generating protein, which may induce tumorigenesis by up-regulating VEGF ((19). Although pioglitazone was shown to inhibit cyclooxygenases-2 (COX- 2), several studies have reported that pioglitazone can also induce increased expression and activity of COX-2 (20)(21)(22). In contrast, reduction in PGE2 levels induced by pioglitazone in lung cancer cells was shown to be through COX-2 independent pathway, without affecting prostaglandin synthases (23). Whether pioglitazone induces reduction of PGE2 through COX-2 dependent or independent pathway, PGE2 can also be induced or produced by several other signaling pathways that are not directly associated with pioglitazone. Increased expression and activation of P2X7R in diabetic bladder urothelium was shown to induce the release of PGE2 and ATP (24) (25) and VEGF signaling have also been shown to be associated with the production of prostaglandins (26). Furthermore, increased production of COX-2-derived prostaglandin I2 (PGI2) has been reported after pioglitazone treatment (27). PGI2 is a hypoxia induced metabolite, which is associated with cancer and other diseases (28). Hypoxia may result in increased expression of COX-2 (29)(30)(31)) and probably pioglitazone treatment induced hypoxia may result in the production of COX-2 derived PGE2 production. Pioglitazone may increase the availability of arachidonic acid, the precursor of production of prostaglandins, without affecting COX-1, COX-2, and cPLA2 expression (32)(33). Increased COX-2 activity, without altering COX-2 expression, was reported in rat myocardium after pioglitazone treatment (34)(35)(36) Thus, elevated levels of PGE2 can be induced by multiple mechanisms irrespective of COX-2 inhibition by pioglitazone. Based on the above-mentioned observations, we believe deregulation of PGE2 may play a critical role in the formation of bladder cancer from pioglitazone treatment. Therefore, PGE2 may be a potential companion diagnostic biomarker for predicting bladder cancer formation in pioglitazone treated patients as well as a theranostics biomarker of bladder cancer. This Fig. 1: be further supported by following pioglitazone (Actos) induced hypothesis canPotential pathways associated with direct and indirect evidences: bladder cancer formation in patients with diabetes 2 | P a g e Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors h t t p : / / w w w . s c i c l i p s . c o m - Analysis using pioglitazone (Actos) and bladder cancer

Fig. 1: Potential pathways associated with pioglitazone (Actos) induced bladder cancer formation in

patients with diabetes

1) Increased production of PGE2 in glomeruli isolated from streptozotocin-induced diabetic rats (37) and in serum from children with diabetes (38) were reported. Elevated monocyte COX-2 expression and circulating PGE2 levels were reported in diabetic patients (39) and increased production of PGE2 was reported in all organs other than kidneys (40) in diabetes. Up-regulation of PGE2 may be associated with pathogenesis of diabetic retinopathy (78). Moreover, PGE inhibitors have shown to improve insulin secretion in type 2 diabetes (41).

3 | P a g e Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors h t t p : / / w w w . s c i c l i p s . c o m - Analysis using pioglitazone (Actos) and bladder cancer

2) PGE2 may activate cancer formation though EP receptors, which can activate EGFR signaling though the activation MMPs. PGE2 signaling stimulate cAMP production through both EP2 and EP4 receptors and promote tumor growth by inhibiting apoptosis (42). EP4 receptor as was reported to be a potential therapeutic target for pancreatic cancer (43)(44)(45). In human nonsmall-cell lung cancer (NSCLC), PGE2 can promote malignant growth by stimulating angiogenesis, tumor invasiveness, apoptosis resistance and inhibition of immune surveillance (46). PGE2 could be a candidate biomarker of bladder transitional cell carcinoma (TCC) (47). 3) Activation of EGFR signaling pathway was shown to be associated with induction of urothelial proliferation over differentiation (48), where EGFR activation can be increased by EGFR ligands such as HB-EGF produced by urothelial cells (49). EGFR signaling also controls PGE2 catabolism through the downregulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme that degrades PGE2, which was also shown to be induced by pioglitazone (50). PGE2 can activate EGFR signaling through EP-receptor and the release of AR from plasma membrane through activation of MMP activity (51). Induction of MMP9 by EGF was reported in bladder cells and MMP9 may be a potential biomarker of bladder cancer (49)(52). PGE2 induces the expression of metalloproteinases by a multistep process involving NF-kappaB (53). 4) Increased expression of VEGF by PGE2 through EP2 and EP4 receptors have been reported (54). 5) Increased secretion of PGE2 was reported to be associated with inhibition of antigenpresenting cell (APC) functions, secretion of Th2 cytokines, promotion of immunosuppressive microenvironment, malignant transformation and tumor progression through local immune suppression (55)(56)(57)(58)(59)(60). Secretion of significant amounts of PGE2 by bladder tumor cells have been reported (61). 6) The arachidonic acid (AA) PGE2 pathway is associated with aggressiveness of human transitional carcinoma (TCC) (62). In prostate cancer cells, arachidonic acid (AA) was shown to induce c-Fos mRNA expression in a PKA-dependent pathway via the EP4 receptor (63). c-Fos was found to be associated with the development of human bladder transitional epithelial cell carcinoma (BTCC) (64). 7) Studies have shown that PGE2 can activate bladder cancer stem cells (65) and this was further confirmed by the expression of stem cell markers Oct3/4 and CD44v6 in bladder cancer (66) (67)(68). CD44v6 was found to be a molecular marker of bladder cancer (69). Therefore, bladder cancer stem cell markers could be used as potential biomarkers for identifying pioglitazone induced bladder cancer formation. 8) Another important observation is the role of COX-2derived PGE2, as well as EGFR signaling, in epithelial cell to a mesenchymal-like phenotype transition or transformation (EMT), which was induced by reduced levels of E-cadherin. Over expression of COX-2 can reduce the levels of E-cadherin through transcriptional repressors ZEB1and Snail (70). Reduced expression of E-cadherin is associated with invasive bladder cancer (71). Moreover, Slug (Snail family of zinc finger transcription factor) plays a role in invasive or 4 | P a g e Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors h t t p : / / w w w . s c i c l i p s . c o m - Analysis using pioglitazone (Actos) and bladder cancer

metastatic bladder cancer and promotion of EMT through cadherin (72). Snail and Slug can be used as biomarkers for predicting tumor recurrence in superficial bladder cancers ((73). Slug may also be used as a potential marker or target for improving the diagnosis and treatment of muscle-invasive bladder cancers (74). Studies have shown that the high mobility group A2 (HMGA2) gene, which was induced by the Smad pathway during EMT (75), can be a potential prognostic marker for predicting tumor recurrence and progression in bladder cancer (76). 9) COX-2/PGE2 signaling can lead to tumor initiation through aberrant activation of betacatenin (80). 10) PGE2 activates phosophoinositide 3-kinase (PI3K) and protein kinase Akt through EP2 receptor, and increased expression of the pro-survival protein Bcl-2, a suppressor of p53 induced apoptosis, via COX-2/PGE2 signaling (81). Activation of PI3K and decreased expression of p53 was shown to be associated with high susceptibility to bladder cancer in type 2 diabetes mellitus model Zucker diabetic fatty (ZDF) rats (82). 11) Wnt-PGE2 interaction regulates vertebrate development and organ regeneration (83) and the deregulated wnt signaling palys a role in urothelial cell carcinoma (UCC) development (84). It is also important to point out that, PGE2 derived from dietary fatty acids may also induce increased PGE2 signaling that may contribute to the onset of cancer. Dietary fatty acids, consisted of essential polyunsaturated fatty acid, linoleic acid, have been shown to be associated with prostate, colon and breast cancer (85). Arachidonic acid (AA) derived from linoleic acid is a precursor of PGE2 and consumption of linoleic acid rich food may result in activating PGE2 signaling pathways. For example, corn consumption may result in increased levels of gastric PGE2 (86) and high linoleic acid diet may result in elevated urinary <a href= (87) or salivary gland and gingival (86) or plasma PGE2 levels (88). Riboflavin deficiency can also lead to increased production of PGE2 (89). It may be interesting to study the food habits and the incidence of bladder cancer in diabetic patients who were treated with pioglitazone (Actos). Moreover, the risk in developing bladder cancer with the consumption of linoleic acid containing foods in combination with pioglitazone (Actos) treatment warranted further investigation. 2. 15-Hydroxyprostagladin dehydrogenase (PGDH) the PGE2 degrading enzyme, was shown to be associated with bladder cancer development and progression (90) and was reported as a potential biomarker of bladder cancer (91). Pioglitazone treatment was shown to increases the production of PGDH (92)(93). Therefore, PGDH could be a potential biomarker, in conjunction with elevated PGE2 levels, for predicting the risk in developing bladder cancer from pioglitazone treatment.

5 | P a g e Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors h t t p : / / w w w . s c i c l i p s . c o m - Analysis using pioglitazone (Actos) and bladder cancer

3. Urinary citrate Calcium-containing urinary solids, resulted from low citrate levels, induced by pioglitazone treatment were found to be associated with increased urothelial cytotoxicity, necrosis, and regenerative proliferation (94). Although increased citrate synthase activity has been reported in pioglitazone-treated patients, urinary citrate was decreased in these patients and induction of microcrystalluria, which may be involved in the development of bladder cancer, have been reported after pioglitazone treatment (95). High dose of pioglitazone (40 microM) was shown to decrease citrate synthase activity in NT2 cells(96). These studies indicate that urinary citrate levels may be a potential biomarker for assessing the risk in developing bladder cancer following pioglitazone treatment. Although the results from our exploratory analysis are hypothetical and may not be conclusive, it is evident from the strategy we have adopted for this analysis that cancer diagnostic biomarkers may be potentially used for developing companion diagnostics tools that can be used for predicting cancer forming side effects of non-anticancer therapeutic drugs. Comprehensive data mining and intelligent hypothesis driven data integration using different cancer specific diagnostic biomarkers and therapeutic drug targets may help in identifying potential companion diagnostic biomarkers, which may lead to the development of clinically viable diagnostic and theranostics tools. Note: This scientific blog is a contribution from Sciclips Consultancy team. References References are hyperlinked to respective abstracts or full articles. Please click the reference numbers to the citation details Related blogs on biomarkers:
Strategies for Rational and Personalized Cancer Biomarker Discovery Cancer Theranostics Potential Applications of Cancer Biomarker Database How to Identify Clinically Successful Biomarkers? Potential Use of Drug Response-Efficacy Biomarkers for Predicting Life-Threatening Disease Causing Side Effects of Therapeutic Drugs Metabolon vs. Stemina Are Biomarkers Patents can be Considered as True Inventions?

Related tools:
Comprehensive cancer biomarker database with companion diagnostics pathway Bioprotocols database

6 | P a g e Cancer Biomarker Strategy to Develop Companion Diagnostics for Predicting Prescription Drug Induced Tumors h t t p : / / w w w . s c i c l i p s . c o m - Analysis using pioglitazone (Actos) and bladder cancer