RASSF1C is a major isoform of the RASSF1 gene, and is emerging as an oncogene. This is in contradistinction to the RASSF1A isoform, which is an established tumor suppressor. We have previously shown that RASSF1C promotes lung cancer cell proliferation and have identified RASSF1C target genes with growth promoting functions. Here, we further report that RASSF1C promotes lung cancer cell migration and enhances lung cancer cell tumor sphere formation. We also show that RASSF1C over-expression reduces the inhibitory effects of the anti-cancer agent, betulinic acid (BA), on lung cancer cell proliferation. In previous work, we demonstrated that RASSF1C up-regulates piwil1 gene expression, which is a stem cell self-renewal gene that is over-expressed in several human cancers, including lung cancer. Here, we report on the effects of BA on piwil1 gene expression. Cells treated with BA show decreased piwil1 expression. Also, interaction of IGFBP-5 with RASSF1C appears to prevent RASSF1C from up-regulating PIWIL1 protein levels. These findings suggest that IGFBP-5 may be a negative modulator of RASSF1C/ PIWIL1 growth-promoting activities. In addition, we found that inhibition of the ATM-AMPK pathway up-regulates RASSF1C gene expression.

RASSF1A has been demonstrated to be a tumor suppressor, while RASSF1C is now emerging as a growth promoting protein in breast and lung cancer cells. To further highlight the dual functionality of the RASSF1 gene, we have compared the effects of RASSF1A and RASSF1C on cell proliferation and apoptosis in the presence of TNF-α. Overexpression of RASSF1C in breast and lung cancer cells reduced the effects of TNF-α on cell proliferation, apoptosis, and MST1/2 phosphorylation, while overexpression of RASSF1A had the opposite effect. We also assessed the expression of RASSF1A and RASSF1C in breast and lung tumor and matched normal tissues. We found that RASSF1A mRNA levels are significantly higher than RASSF1C mRNA levels in all normal breast and lung tissues examined. In addition, RASSF1A expression is significantly downregulated in 92% of breast tumors and in 53% of lung tumors. Conversely, RASSF1C was upregulated in 62% of breast tumors and in 47% of lung tumors. Together, these findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor but instead may play a role in stimulating survival in breast and lung cancer cells.

Introduction: We have previously shown that RASSF1C promotes cell proliferation, migration and attenuates apoptosis in cancer cells. We have also shown that RASSF1C is significantly up-regulated in breast and lung cancer tissues compared to normal tissues. Together these demonstrate that RASSF1C, is not a tumor suppressor like RASSF1A, but instead appears to function as an oncoprotein. Currently nothing is known about the upstream signaling cascades involved in regulating RASSF1C gene expression and thus we performed transcriptome PCR array study to identify chemicals inhibitors that modulate(s) RASSF1C expression. Method: A transcriptome PCR array was obtained from Qiagen. It consisted of cDNA from breast cancer cell line MCF7 cells treated with 90 different chemical inhibitors that regulate various signaling pathways. The array was screened with RASSF1C gene specific primers. Data analysis was performed by importing the Ct values obtained into data analysis software. Results: The PCR array screen identified several chemical reagents that appear to up-regulate and several that seem to down-regulate RASSF1C expression by ≥ 1.5 fold. The two most notable reagents are Dorsopmorhin (AMPK inhibitor) which up-regulates RASSF1C expression by 2.8 fold and Trichostatin A (HDAC inhibitor) which down regulates RASSF1C expression by 2 fold. We are in process of confirming the effect of these inhibitors in a panel of breast and lung cancer cells. Conclusion: The findings are novel and suggest that inhibition of AMPK pathway induces RASSF1C expression while inhibition of HDAC pathway suppresses RASSF1C gene expression. We should note that Trichostatin A has been shown to activate AMPK, inhibits HDAC, arrests cell growth, and induces apoptosis in human cancers. Our findings provide a potential mechanism for regulating RASSF1C gene expression through the modulation of AMPK and HDAC pathways. Citation Format: Yousef G. Amaar, Mark E. Reeves, Matthew Firek. Ampk and hdac pathways and regulation rassf1c gene expression. [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 2454. doi:10.1158/1538-7445.AM2014-2454

Introduction: Recently, RASSF1A has been shown to mediate the apoptotic effects of TNF-α by interacting with the mammalian sterile 20-like kinase 1 and 2 (MST1/2) through the Salvador/Rassf/Hippo (SARAH) domain leading to MST1/2 phosphorylation activation of apoptosis through the Hippo pathway. In contrast, we have previously shown that RASSF1C promotes cell proliferation and attenuates apoptosis in cancer cells. Since both RASSF1A and RASSF1C contain the SARAH domain located in their identical C-termini, RASSF1C should be capable of interacting with SARAH domain-containing proteins, and could potentially attenuate MST1/2-mediated apoptosis through the Hippo pathway. Thus, in this study we have investigated the impact of RASSF1C on MST1/2 activation/phosphorylation in presence of TNF-α, including whether RASSF1C modulates MST1/2 pro-apoptotic effects through the Hippo pathway differently from RASSF1A. Method: Breast and lung cancer cells over-expressing RASSF1A and RASSF1C were used in this study. Cells were cultured in the proper media and treated with TNF-α for 12-18 hr. Cells were collected and used for Western blot analysis utilizing antibodies that detect MST and p-MST antibodies. Results: Our preliminary data suggest that RASSF1C over-expression decreases the phosphorylation levels of MST1/2 in TNF-α-treated breast and lung cancer cells compared to those cells over-expressing RASSF1A Conclusion: Our findings suggest that over-expression of RASSF1C in breast and lung cancer cells may attenuate the MST1/2 apoptotic effects by sequestering of MST1/2 proteins and inhibiting their activation by phosphorylation. The findings also further support our hypothesis that RASSF1C attenuates apoptosis; and RASSF1C, unlike RASSF1A, may inhibit MST1/2 activation and hence may negatively modulate the Hippo pathway. Citation Format: Yousef G. Amaar, Matthew Firek, Mark E. Reeves. RASSF1C, unlike RASSF1A, reduces TNF-α induced phosphorylation of MST1/2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4288. doi:10.1158/1538-7445.AM2013-4288

Introduction: RASSF1C is emerging as an important oncoprotein in lung cancer cell growth. We have shown that RASSF1C promotes lung cancer cell proliferation and migration; and RASSF1C up-regulates important genes in lung cancer cell growth that include a stem cell self-renewal gene, piwi1 (hiwi). PIWI-like proteins are a subfamily of Argonaute proteins that interact with small PIWI-interacting RNA molecules (known as piRNAs that are 24-32 nucleotides long) to form complexes that regulate transcriptional and translational repression. This leads to inhibition of apoptosis, stimulation of cell division and proliferation, and down-regulation of cyclin inhibitors and tumor suppressors. Therefore, modulation of Piwil1-piRNA gene expression by RASSF1C suggests a potential role for RASSF1C in lung cancer stem cell development and progression. To further investigate our hypothesis, we carried out a global piRNA microarray screen to identify piRNAs that are modulated by RASSF1C in lung cancer cells. Method: A piRNA microarray screen was performed using the lung cancer cell line H1299 stably over-expressing RASSF1C (and controls). Total RNA was extracted from experimental and control cells and was submitted to Arraystar (Rockville, MD) for the piRNA microarray screen and data analysis. Results: The piRNA microarray screen identified several piRNAs that are regulated by RASSF1C and we have confirmed the expression of some of them in cell lines. The function of the piRNAs identified is yet to be determined. We have initiated studies to determine the function of some of the most up-regulated and down-regulated piRNA genes in lung cancer cells. We are also profiling the expression of these piRNAs in normal and lung tumor tissues. Conclusion: Several piRNAs are target genes of RASSF1C. Characterization of the function of these piRNAs may enhance our understanding of the role of RASSF1C in promoting lung cancer stem cell growth and progression. Linking a Rassf1c-Piwil1/piRNAs axis to lung cancer stem cell development and progression could possibly lead to discovery of new diagnostic and therapeutic targets for lung cancer. Citation Format: Yousef G. Amaar, Matthew Firek, Mark E. Reeves, Mark E. Reeves. RASSF1C modulation of Piwi-interacting RNAs (piRNAs) in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2146. doi:10.1158/1538-7445.AM2015-2146