Bhawana Dikshit

FAT1 acts as an upstream regulator of oncogenic and inflammatory pathways, via PDCD4, in glioma cells

Glioblastoma multiforme (GBM) is the most aggressive and the commonest primary brain tumor with a tendency for local invasiveness. The pathways of neoplasia, invasion and inflammation are inextricably linked in cancer and aberrations in several regulatory pathways for these processes have been identified. Here we have studied the FAT1 (Homo sapiens FAT tumor-suppressor homolog 1 (Drosophila)) gene to identify its role in the tumorigenecity of the gliomas. The expression of FAT1 was found to be high in grade IV glioma cell lines (U87MG, A172, U373MG and T98G) but low in grade III glioma cell lines (GOS3 and SW1088). Two cell lines (U87MG and A172) with high FAT1 expression were chosen for in vitro FAT1-knockdown studies. FAT1 knockdown by small interfering RNA resulted in decreased migration and invasion of both the cell lines along with increased expression of the tumor-suppressor gene programmed cell death 4 (PDCD4). Increased PDCD4 expression led to the attenuation of activator protein-1 (AP-1) transcription by inhibiting c-Jun phosphorylation and resulted in concomitant decrease in the expression of AP-1-target genes like MMP3, VEGF-C and PLAU, the pro-inflammatory regulator COX-2 and cytokines IL1β and IL-6. Conversely, simultaneous silencing of PDCD4 and FAT1 in these cells significantly enhanced AP-1 activity and expression of its target genes, resulting in increase in mediators of inflammation and in enhanced migratory and invasive properties of the cells. We also observed a negative correlation between the expression of FAT1 and PDCD4 (P=0.0145), a positive correlation between the expression of FAT1 and COX-2 (P=0.048) and a similar positive trend between FAT1 and IL-6 expression in 35 primary human GBM samples studied. Taken together, this study identifies a novel signaling mechanism mediated by FAT1 in regulating the activity of PDCD4 and thereby the key transcription factor AP-1, which then affects known mediators of neoplasia and inflammation.

A Combined Gene Signature of Hypoxia and Notch Pathway in Human Glioblastoma and Its Prognostic Relevance

Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor—clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.

FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia‐mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significance of the FAT1‐HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1α and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion‐associated reduction in HIF1α was due to compromised EGFR‐Akt signaling as well as increased VHL‐dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1‐HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

FAT1 (FAT tumor suppressor homolog 1 (Drosophila))

Review on FAT1 (FAT tumor suppressor homolog 1 (Drosophila)), with data on DNA, on the protein encoded, and where the gene is implicated.

PO-125 FAT1 on salvador-warts-hippo (SWH) pathway in human glioblastoma

Introduction Glioblastoma(GBM) is an aggressive brain tumour arising from glial cells. Our lab has identified the oncogenic role of FAT1 gene in GBM, regulating inflammatory and hypoxic microenvironment of the tumour as well as migratory/invasive properties of the tumour cells. In Drosophila, fat, the ortholog of FAT1, is known to regulate the Salvador-Warts-Hippo (SWH) pathway, but its role in human is not clear. Here, we have analysed the effect of FAT1 on SWH pathway in glioma. Material and methods Glioma cell lines (U87MG, U373, A172, GOS3 and SW1088) were transfected with FAT1 specific siRNA/control siRNA and analysed the expression of SWH pathway molecules by qPCR/Western blot. Protein-protein interactions were analysed by Co-immunoprecipitation (Co-IP) after overexpression of YAP1 (wild-type and mutated) and TEAD1 with and without FAT1 knockdown. Sub-cellular localization of proteins was analysed by Confocal microscopy. Results and discussions The mRNA expression of FAT1 and SWH pathway molecules (MST1, LATS1, LATS2, YAP1 and TEAD1) was highest in U87MG cells followed by A172, U373MG and GOS3. After FAT1 knockdown, the mRNA expression of MST1 and BIRC2 were significantly decreased with no change in the levels of LATS1, LATS2, YAP1, TEAD1 and BIRC5. At protein level, increased YAP1 and phospho-YAP1 was observed after FAT1 knockdown with increased total as well as phospho-YAP1 in the cytoplasmic extract as compared to the nuclear extract. There was significant reduction in the interaction between YAP1 and TEAD1 in siFAT1 treated cells as compared to siControl treated cells. Conclusion Knockdown of FAT1 (i) increases the YAP1 protein level, could be by increasing the protein stability as no change was observed at the mRNA level (ii) it relieves the inhibitory effect on YAP1 phosphorylation, thereby increasing the phospho-YAP1 level (iii) it affects the sub-cellular localization of YAP1 by retaining YAP1 in the cytosol and thereby (iv) decrease in the YAP1:TEAD1 interaction with decreased expression of their target gene Birc2. This finding of the effect of FAT1 on YAP1 in GBM is novel with features pointing towards the oncogenic role of FAT1 by regulating YAP1 sub-cellular localization and co-transcriptional activity independent of SWH pathway.

Abstract 988: Triple negative breast cancer cells have higher dependence on mitophagy for their survival as compared to hormone positive breast cancer cells

Autophagy is a key pathway utilised by the cells to clear the dysfunctional cellular components and maintain homeostasis. However cancer cells having defective apoptosis, deploy autophagy as a survival mechanism by limiting tumor necrosis and metabolic stress. Autophagy has been reported to be upregulated in triple negative breast cancers which are categorized as the most aggressive breast cancers with poor prognosis. Nevertheless the exact mechanism of autophagic upregulation in TNBC is still elusive. Therefore analyzing pathways specifically altered in these tumors might provide better targets for therapeutic intervention. Results: In the present study we observed higher basal autophagy in TNBC cell line MDA MB 231 as compared to triple positive MCF7 cells as analyzed by the presence of higher number of GFP-LC3 puncta in the former. Treatment with 20μM of autophagy inhibitor chloroquine for 48 hrs led to significant cell death in TNBC cell line as compared to MCF7 whereas treatment with 20μM of PI3K inhibitor wortamannin did not significantly affected the survival of TNBC and MCF7 cells. Further the analysis of autophagy markers revealed higher LC3B and Beclin1 levels as compared to ATG5 and ATG7 levels in MDA MB 231 cells both at mRNA and protein levels as analyzed by qPCR and western blot. Also the knockdown of ATG7 by ATG7 shRNA did not significantly affected the survival of TNBC cells. Conclusion: Thus in this study we aim to highlight the difference in the expression of autophagy markers and the effect of autophagy inhibitors wherein only late phase of autophagy seems to significantly upregulated in TNBC. The difference can be attributed to the dependence of TNBC cells upon ‘late phase autophagy’ or particularly ‘mitophagy’ that is crucial for rapid clearance of misfolded proteins and dysfunctional mitochondria as has been reported in TNBCs. Further study to dissect the mechanism of selective upregulation of ‘late phase autophagy’or ‘mitophagy’ in TNBC might provide novel targets for treatment strategies in these breast cancers. Citation Format: Bhawana Dikshit, Aakansha Rai, Soumya Sinha Roy. Triple negative breast cancer cells have higher dependence on mitophagy for their survival as compared to hormone positive breast cancer cells. [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 988. doi:10.1158/1538-7445.AM2015-988

Nuclear Factors Linking Cancer and Inflammation

An association between inflammation and cancer has long-been known, but the past decade has witnessed a spurt in the research linking the two processes. On the one hand chronic inflammation predisposes to cancer, on the other, neoplastic transformation predisposes towards an intrinsic pro-inflammatory microenvironment, which further promotes the progression of the malignancy. Irrespective of the stimulus, whether extrinsic (bacteria, viruses, non-healing wounds, irritants etc.) or intrinsic (oncogenes, protein kinases etc.), all signals that trigger the inflammatory microenvironment in tumor cells converge in the nucleus and coordinate inflammatory transcriptional activity by activating various nuclear/transcription factors. This forms a vicious cycle, further promoting inflammation, facilitating tumor progression, proliferation, survival and angiogenesis. This chapter focuses on transcriptional mediators intrinsic to tumour cells that enhance the inflammatory processes.

Abstract 4102: FAT1: A novel regulator of cancer and inflammation.

FAT1, a member of cadherin superfamily is a large trans-membrane protein with 34 cadherin repeats. FAT1 was originally identified as a tumor suppressor gene in Drosophila but its role in human cancers is just beginning to unfold. Considering its tumor suppressive role in Drosophila, few studies have reported similar function of FAT1 in human cancers but on the contrary several studies have also demonstrated oncogenic role of FAT1. In this study we identified oncogenic role of FAT1 in human glioma where knockdown of FAT1 was found to inhibit migration and invasion of glial cells. Further investigation into molecular mechanism of FAT1 function revealed direct link to AP-1 mediated transcription via Programmed Cell Death 4 (PDCD4). Knockdown of FAT1 was found to inhibit AP-1 mediated transcription and expression of downstream target genes. AP-1 is a known mediator of cancer and inflammation and accordingly we found decreased expression of several pro-inflammatory markers like COX-2, cytokines like IL-6, IL-1β and chemokines like IL-8, MCP-1 and MIP-2 after FAT1 knockdown. Interestingly, the expression of anti-inflammatory cytokine IL-10 was found to be increased after FAT1 knockdown. Moreover FAT1 expression was also found to positively correalate with COX-2 and IL-6 expression in primary GBM tumors. Thus, in this study FAT1 was found to act as an oncogene as well as to orchestrate the inflammatory milieu of the glioma cells by regulating AP-1 mediated transcription via PDCD4. Citation Format: Bhawana Dikshit, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1: A novel regulator of cancer and inflammation. [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 4102. doi:10.1158/1538-7445.AM2013-4102