Abstract 1937: RBM3 overexpression increases β-catenin signaling activity to induce a cancer stem cell phenotype
Authors: Venugopal Anand , Balmaceda Julia , Kwatra Deep , Subramaniam Dharmalingam , Umar Shahid , Anant Shrikant 
Published: May 2015

Colon cancer is the second leading cause of cancer related deaths in the US. We have previously demonstrated that the RNA binding protein RBM3 is oncogenic when overexpressed in NIH 3T3 cells. Furthermore, several studies have shown that RBM3 expression increases in high grade cancer compared to lower grade cancer or normal adjacent tissues. While a correlation between RBM3 expression and the progression of malignancies is apparent, the evidence implying a causative link is lacking. Here we investigate the effects of exogenous RBM3 overexpression on the phenotype of colon cancer cell lines HCT 116 and DLD-1. We developed cells with stable doxycycline (Dox) inducible RBM3 overexpression. Upon RBM3 overexpression, there was an increase in the percentage of Hoescht 33342 effluxing side population cells and spheroid formation capacity, both of which are populations with increased capacity to self-renew and differentiate. Additionally, we observed that RBM3 overexpression increases CD44 protein levels. These studies imply that RBM3 overexpression increases colon cancer stem cell phenotype. To elucidate the mechanism by which RBM3 overexpression increases the stem cell phenotype, we next investigated the β-catenin signaling pathway. The majority of colon cancers initiate their malignant transformation with an activating mutation of the Wnt/β-catenin signaling pathway which is critical in maintaining the stem cells within the intestinal epithelium. The small pool of cytoplasmic β-catenin is normally degraded, however, upon inhibition of the destruction complex, it translocates to the nucleus and induces transcriptional changes within the cell. RBM3 overexpression showed increase in both total and nuclear β-catenin in the two cell lines. To demonstrate directly that β-catenin is transcriptionally active, we used the TOPFlash luciferase reporter system. There was a significant increase in luciferase activity in RBM3 overexpressing cells confirming the increased nuclear β-catenin levels. Furthermore, there was increase in the expression of c-Myc, CD44 and LGR5 mRNA, all transcriptional targets of β-catenin. In previous studies, we have demonstrated that RBM3 induces the expression of cyclooxygenase-2 and interleukin-8, both of which can act through AKT to inhibit GSK3β, a member of the β-catenin destruction complex. Therefore, we next investigated effects of RBM3 overexpression on GSK3β activity. There was a significant increase in activating phosphorylation at Akt at Ser473, and inhibitory phosphorylation of GSK3β at Ser9. Additionally, β-catenin phosphorylation at Ser33/37/Thr41, targets of GSK3β was marked lower. Taken together, these data suggest that when overexpressed, RBM3 induces β-catenin signaling to increase stem cell features in colon cancer cells. Additionally, these data show a novel causative link between RBM3 levels and the progression of colon cancer.

Citation Format: Anand Venugopal, Julia Balmaceda, Deep Kwatra, Dharmalingam Subramaniam, Shahid Umar, Shrikant Anant. RBM3 overexpression increases β-catenin signaling activity to induce a cancer stem cell phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1937. doi:10.1158/1538-7445.AM2014-1937