Identification of Novel Pathways that Promote Anoikis through Genome-wide Screens

Epithelial cells that lose attachment to the extracellular matrix (ECM) undergo a specialized form of apoptosis called anoikis. Anoikis has an important role in preventing oncogenesis, particularly metastasis, by eliminating cells that lack proper ECM cues. The basis of anoikis resistance remains to be determined and to date has not been linked to alterations in expression or activity of previously identified anoikis effector genes. Here, I utilized two different screening strategies to identify novel anoikis effector genes and miRNAs in order to gain a deeper understanding of anoikis and the potential mechanisms of anoikis resistance in cancer. Using large-scale RNA interference (RNAi) screening, I found that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. I found that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis I show that knockdown of Kdm3a enhances metastatic potential. Finally, I find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, my results reveal a novel transcriptional regulatory program that mediates anoikis. Next, I sought to discover miRNAs involved in anoikis by investigated changes in miRNA expression during anoikis using small RNA sequencing technology. Through this approach I discovered that miR-203 is an anoikis effector miRNA that is also highly down-regulated in invasive breast cancer cells. In breast epithelial cells, miR-203 is induced upon the loss of ECM attachment and inhibition of miR-203 activity leads to a resistance to anoikis. I utilized a dual functional- and expression- based RNA sequencing approach and found that miR-203 directly targets a network of pro-survival genes to induce cell death upon detachment. Finally, I found that the loss of miR-203 in invasive breast cancer leads to the elevation of several anoikis-related pro-survival target genes to contribute to anoikis resistance. Taken together, my studies reveal novel pathways through which cell death is induced upon detachment from the ECM and provide insight into potential mechanisms of anoikis resistance in cancer

RNA-Sequencing Reveals Direct Targets of Tumor Suppressor miR-203 in Human Mammary Epithelial Cells

Background: Breast cancer is the leading cause of cancer-related mortality in women worldwide. Since a significant portion of cases present with or progress to metastatic disease, furthering our understanding of metastasis is critical to develop better treatments. Epithelial cells maintain contact with the extracellular matrix (ECM) predominantly via integrin engagement, a process required for tissue integrity and barrier function. In non-transformed cells, loss of ECM adhesion promotes a specialized form of programmed cell death, anoikis. In order for efficient metastasis to occur, breast tumor cells must evade anoikis. miR-203, known to be down-regulated in several cancers, was found by our lab to be induced ten-fold 24 hours following detachment in breast epithelial cells, but not invasive triple negative breast cancer (TNBC) cells, suggesting that miR-203 may participate in promoting anoikis. Interestingly, more invasive breast cancer cell lines have been shown to express miR-203 at significantly lower levels than those of less invasive lines. Objectives: Since restoration of miR-203 expression ectopically is not feasible in a clinical setting, we sought to identify and characterize miR-203 target genes in order to provide a pharmaceutical platform for restoration of anoikis sensitivity in metastatic breast cancer. Methods: We performed traditional RNA-sequencing (RNA-Seq) coupled with immunoprecipitation of the RNA-induced silencing complex (RISC; Ago2 RIP-Seq) in MCF-10A, an immortalized, but non-transformed breast epithelial cell line, overexpressing precursor miR-203 or an empty vector control. MDA-MB-231, triple negative ductal carcinoma cells, were used as our invasive comparison cell line. Results: Here we show that miR-203 induction in detached MCF-10A cells is due to loss of integrin signaling. Our coupled RNA-Seq and Ago2 RIP-Seq approach revealed 72 potential candidates, 42 of which were predicted miR-203 targets based on the TargetScan algorithm. We subjected the candidates to stringent characterization and found 9 bona-fide miR-203 targets that promote cell death when inhibited. Among these, WDR69, PRKAB1, PRPS2, and HBEGF were significantly elevated in TNBC tumor samples, as determined by RNA-Seq analysis in The Cancer Genome Atlas (TCGA). Conclusion: Understanding the mechanisms by which cells evade anoikis during tumor dissemination is crucial to developing more effective therapies in breast cancer. miR-203, which is expressed at very low levels in more invasive breast cancers, is a positive regulator of anoikis that is upregulated in response to loss of contact with the ECM. Our combined RNA-sequencing screen revealed 42 direct miR-203 targets. Inhibition of 9 bona-fide targets promoted cell death, suggesting that they are negative regulators of anoikis. WDR69, PRKAB1, PRPS2, and HBEGF were all significantly elevated in TNBC tumor samples relative to less invasive samples, likely a consequence of low miR-203 expression. The identified genes represent potential pharmaceutical targets for novel breast cancer therapies

The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L

Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis

Reviewer Database and Evaluation Process

Effective reviewer selection at the National Science Foundation can make the review process, the funding decision, and the communication with Principal Investigators more successful. The purpose of our project was to create and validate a process for evaluating panel reviewers and create a searchable and updateable reviewer database. We conducted interviews and surveys of Program Directors to gather and analyze information for developing the evaluation tool and the database, and performed validation testing of our tool