Despite recent advances in therapeutics, colorectal cancer remains the third deadliest cancer in the USA. This is mainly attributable to survival of a small population of cancer cells called stem cells (CSCs) with the ability to self-renew, resist chemotherapy killing and metastasize (broadly speaking CSC phenotype). We have generated cells resistant to a combination of 5-flurouracil (5-FU) and oxaliplatin (FUOX), the backbone of colorectal cancer chemotherapeutics. These FUOX-resistant cells are enriched in CSCs and show increased expression of total and activated form of IGF-1R as well as its ligand IGF-2. Inhibition of IGF-1R by a tyrosine kinase inhibitor, GSK1904529A, results in a dose dependant decrease in colonosphere formation (a measure of CSCs growth) which is especially pronounced in p53 wild type cells. Moreover, combination of FUOX with GSK1904529A results in not only synergistic inhibition of primary colonosphere formation but also secondary sphere formation suggesting IGF-1R may play a role in CSCs self-renewal. MicroRNAs (miRs) have been identified as important negative regulators of gene expression in embryonic and cancer stem cell growth. We hypothesized that IGF-1R inhibition would lead to a change in expression of specific miRs. Indeed, we identified miR-363, a non p53 regulated miR, and miR-215, a p53 regulated miR, to be highly overexpressed (28-fold and 6-fold respectively) following IGF-1R inhibition. More importantly, we found that expression of miR-363 and -215 is decreased several fold in FUOX-resistant cells that are enriched in CSCs. At the same time, expression of thymidylate synthase (TS), the putative target of miR-215, as well as integrin alpha-V (CD51) and myeloid cell leukemia-1 (MCL-1), both targets of miR-363 and regulator of CSC phenotype, were highly overexpressed in FUOX-resistant cells. Moreover, both miRs and their respective targets can be favorably modulated by IGF-1R inhibition. Based on the above observation we hypothesize, that IGF-1R inhibition alters the cancer stem cell phenotype thus enhancing effectiveness of chemotherapy in FUOX-resistant colorectal cells. We further hypothesize that this enhanced efficacy is due, in part, to induction of p53 dependent (miR-215) and independent (miR-363) microRNAs. To test this hypothesis, we will examine the effect of IGF-1R inhibition in primary human colon cancer cells (propagated as colonospheres) on CSC phenotype in vitro and the role of miR-215 and miR-363 in the process (Aim 1). We will also test the effect of IGF-1R depletion/inhibition on tumor formation and xenograft growth (with and without FUOX) in vivo (Aim 2). Lastly, we will delineate the role of specific miR-215 (TS and ABCG2) as well as miR-363 (CD51 and MCL-1) target genes in modulating CSC phenotype (Aim 3). The results from the proposed experiments would expand our understanding of mechanisms of colon CSCs growth and chemotherapy resistance resulting in a paradigm shift in treatment of colorectal cancer improving outcome for this deadly disease.
Inhibition of IGF-1R: A Therapeutic Strategy for Colon Cancer Stem-like Cells: Bhaumik B. Patel (PD/PI) Project Narrative and Significance to VA Patient Population Approximately one-third of the veterans are now around 65 years of age or older. Colorectal cancer, the incidence of which increases exponentially with aging, is the third deadliest cancer in the US. The high mortality is attributed to disease relapse which is being attributed to cancer stem cells (CSCs) which represent a small fraction of the tumor but are hard to kill by conventional chemotherapies. Hence targeting CSC would reduce poor treatment outcomes. To this end, we have indentified IGF-1R pathway to play an important role in survival of CSCs. The primary goal of this investigation is to examine IGF-1R inhibition as a therapeutic strategy for selectively eliminating colon cancer CSCs. In addition, we plan to study potential mechanisms with particular reference to two of the IGF-1R regulated microRNAs-215 and -363 and genes regulated by them. If encouraging results from the proposed preclinical study are subsequently confirmed in the clinic, the selective targeting of CSCs will offer a new paradigm in colon cancer diagnostics and therapeutics.
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