Browsing by UMass Chan Affiliation "Molecular, Cell, and Cancer Biology"
Now showing items 1-4 of 4
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Death is Not the End: The Role of Reactive Oxygen Species in Driving Apoptosis-induced ProliferationApoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer growth and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial Drosophila tissues, the Caspase-9 homolog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Using a model of sustained AiP that produces a hyperplastic phenotype in Drosophila eye and head tissue, I have found that caspase-induced activation of JNK during AiP depends on extracellular reactive oxygen species (ROS) generated by the NADPH oxidase Duox. I found these ROS are produced early in the death-regeneration process by undifferentiated epithelial cells that have initiated the apoptotic cascade. I also found that reduction of these ROS by mis-expression of extracellular catalases was sufficient to reduce the frequency of overgrowth associated with our model of AiP. I further observed that extracellular ROS attract and activate Drosophila macrophages (hemocytes), which may in turn trigger JNK activity in epithelial cells by signaling through the TNF receptor Grindelwald. We propose that signaling back and forth between epithelial cells and hemocytes by extracellular ROS and Grindelwald drives compensatory proliferation within the epithelium, and that in cases of persistent signaling, such as in our sustained model of AiP, hemocytes play a tumor promoting role, driving overgrowth.
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Identification of a myotubularin-related phosphatase that regulates autophagic flux and lysosome homeostasisMacroautophagy (autophagy) is a vesicle trafficking process that targets cytoplasmic cargoes to the lysosome for degradation and underlies multiple human disorders. Pioneering work in Saccharomyces cerevisiae defined the core autophagy machinery, but animals possess autophagy regulators that were not identified in yeast. Autophagic flux occurs when autophagy rate increases or decreases in response to various cellular cues, such as nutrient availability. Indeed, dysregulated autophagy rates contribute to disease, making autophagy- modulation a therapeutic avenue to treat cancer, neurodegenerative disorders, and other diseases. To identify novel regulators of autophagy in animals, I investigated autophagy in the context of animal development using Drosophila. In my dissertation, I screened for phosphoinositide phosphatases that influence autophagy, and identifed CG3530/dMtmr6, a previously uncharacterized phosphatase. CG3530/dMtmr6 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphoinositide phosphatases. I showed that dMtmr6 functions as a regulator of autophagic flux in multiple Drosophila cell types, and the MTMR6 family member MTMR8 functions similarly in autophagy of higher animal cells. Decreased dMtmr6 function resulted in autophagic vesicle accumulation, lysosome biogenesis, and impaired both fluid phase endocytosis in the fat body and phagocytosis in embryonic macrophages. Additionally, dMtmr6 is required for development and viability in Drosophila. In human cells, lysosome homeostasis requires both the MTMR8 PH domain and catalytic cysteine residue, but only the PH domain is required to maintain autophagic flux. Collectively, this work identified a role for dMtmr6 and MTMR8 in autophagic flux and lysosome homeostasis.
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Identification of Novel Pathways that Promote Anoikis through Genome-wide ScreensEpithelial 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.
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Investigating the role of the c-Jun NH2-terminal kinase pathway in ErbB2-driven breast cancer and macrophage polarizationBreast cancer is the second most common malignancy in the world, accounting for over 1.7 million new diagnoses and an estimated 500,000 deaths per year (1). Overexpression of the receptor tyrosine kinase ErbB2, also known as Her2 or Neu, occurs in over 30% of breast cancers and correlates with metastasis, poor prognosis, and decreased survival (1, 2). Although therapeutics targeting ErbB2 show clinical efficacy, many patients display no initial response or develop drug resistance over time (2). A deeper understanding of the molecular basis of ErbB2-driven tumorigenesis is thus required for the development of improved therapeutic strategies. In vitro experiments suggest that activation of the c-Jun NH2-terminal kinase (JNK) pathway, a mitogen-activated protein kinase pathway, promotes proliferation, cellular invasion, and stem cell expansion in ErbB2-driven breast cancer (3, 4). Furthermore, unpublished data from our lab using mammary epithelial cells expressing activated ErbB2 show that JNK is required for acinus formation in in vitro 3D cultures. In contrast to these studies showing a tumorigenic role for the JNK pathway, other data from our lab show that JNK loss results in accelerated breast tumor growth, suggesting a tumor suppressive role (5, 6). However, these studies were performed in p53 knockout mice with or without a Kras mutation, where the latter required extensive aging and genomic instability to occur before differences in tumor growth were observable. To date, limited in vivo studies exist to confirm the role of JNK in more biologically relevant breast tumor models, such as in ErbB2-mediated cancer, which accounts for over 30% of all human breast cancers. In addition, the molecular mechanisms by which JNK signaling promotes ErbB2-driven tumorigenesis remains poorly understood. To address the discrepancy in JNK function between the in vitro ErbB2-driven breast cancer data and the in vivo p53 knockout tumor data, I began the development of an in vivo murine model to confirm the role of JNK in ErbB2-driven breast cancer. This mouse model will also allow us to test a potential mechanism by which JNK regulates tumorigenesis. Studies show that ErbB2-mediated secretion of the inflammatory cytokine IL6 promotes transformation and tumor growth by activation of the STAT3 transcription factor, triggering an IL6/STAT3 autocrine signaling loop (7,8). A major regulator of Il6 gene expression includes activator protein 1 (AP-1), a transcription factor composed of downstream JNK targets in the Jun protein family (9). In vitro experiments using ErbB2-overexpressing mammary epithelial cell lines show that chemical inhibition of JNK suppresses secreted IL6 protein levels, supporting a role for the JNK pathway in IL6 regulation (7). Thus, I hypothesize that JNK drives ErbB2-driven breast cancer by promoting IL6-mediated tumor progression. Addressing this will increase our understanding of the role of JNK in ErbB2-driven breast cancer and reveal a potentially new mechanism by which JNK functions in tumor progression. Additionally, I began the development of a mouse model that will allow us to investigate the role of JNK in macrophage polarization as an alternative mechanism by which JNK regulates ErbB2-driven breast cancer. In addition to promoting STAT3-dependent tumor growth, IL6 can indirectly drive tumorigenesis by promoting expression of the IL4 receptor in macrophages, triggering STAT6-mediated macrophage polarization towards the pro-tumorigenic M2 phenotype (10, 11). Unlike classically activated M1 macrophages, which promote inflammation and anti-tumor immunity, alternatively activated M2 macrophages function in immunosuppression and metastasis and correlate with advanced stages of breast cancer (12, 13). Further evidence supporting a role for the JNK pathway in macrophage polarization includes a recent study suggesting that JunB, a downstream JNK target and component of the AP-1 complex, plays a crucial role in the induction of M2 macrophage polarization in human alveolar macrophages (13). I hypothesize that activation of the JNK signaling pathway induces IL6-dependent macrophage polarization towards the pro-tumorigenic M2 phenotype. Addressing this hypothesis will determine for the first time whether JNK functions in regulating macrophage polarization within the tumor microenvironment, offering a potentially new mechanism by which JNK can promote ErbB2-driven breast cancer. Determining the role of JNK in ErbB2-mediated breast cancer will have direct therapeutic relevance, as targeting JNK has the potential to inhibit ErbB2-driven breast cancer and other IL6-mediated diseases. Investigating the underlying mechanisms by which JNK functions in ErbB2-positive breast cancer can also offer new molecular targets and further contribute to effective drug design.
