Wilson disease (WD) is an autosomal recessive disorder characterized by deposition of copper in the body, mainly in the liver and brain. WD patients present with hepatic, neurological, and psychiatric problems. The diagnosis of WD is very challenging, and is performed by taking into account both clinical and biochemical parameters. The treatment of WD exists, which aims at initial chelation therapy followed by maintenance therapy. WD is caused by mutations in the ATP7B gene. Till date, more than 600 mutations in ATP7B have already been described from many countries, including India. However, there are a very few large cohort studies which are reported from Indian population. In this study, we have attempted to perform mutation analysis of ATP7B in a large cohort of WD families from Bangalore, south India, and further look into the molecular consequences of the novel mutations identified in the present study.

Transforming growth factor-β (TGF-β) superfamily of cytokines comprises of several members, which can broadly be sub-divided into three classes [TGF-βs, Activin/Nodal, and Bone morphogenetic proteins (BMPs)]. Most members of this family play critical roles during embryo development differentiation and regulation of homeostasis. In mammals there are three TGF-β isoforms, TGF-β1, 2 and 3. All the three TGF-β isoforms have important roles in embryo development as revealed by mouse knock-out models. TGF-β has also been associated with several pathological conditions such as inflammation, Fibrosis, and cancer. In cancers, TGF-β plays both tumor suppressive and tumor promoting roles depending upon the context. TGF-β has growth inhibitory effect on epithelial cells which is essential to maintain tissue homeostasis. TGF-β induces the expression of several cyclin dependent kinase inhibitors such as p21Cip1, p15Ink4b while down-regulating the expression of cMYC in the epithelial cells. In lieu of its tumor suppressive role, several cancers harbor mutations in the components of the TGF-β signaling axis such as receptors and effector molecules called SMADs. Interestingly various cancers also show hyper activation of TGF-β signaling. It has been suggested that cancer cells become unresponsive to the growth inhibitory effects of TGF-β by losing the expression of p21Cip1, and p15INK4b. Oncogenic transformation of cancer cells can override the growth inhibitory effects of TGF-β. While the loss of growth inhibitory effects by TGF-β are seen in the tumor cells, several tumor promoting actions are also observed in these cells such as induction of EMT. TGF-β activates mesenchymal cells leading to the formation of a reactive stroma in tumors and TGF-β suppresses almost all types of cells of the immune system causing a local immune-suppressive environment. TGF-β also recruits mesenchymal stem cells into the stroma which secrete several cytokines. The sum total of all these effects is pro-angiogenic, pro-infiltrative and pro-metastatic. In the canonical TGF-β signaling pathway, ligands bind to the hetero-tetrameric receptor complex of TGFβR1 and TGFβR2 leading to activation of the TGFβR1 by TGFβR2. Activated TGFβR1 then phosphorylates and activates R-SMAD molecules (SMAD2, SMAD3) which complexes with the co-SMAD (SMAD4) and translocate into the nucleus to effect transcriptional changes. Non-canonical TGF-β signals are many and almost all the known signaling pathways like MAPK, WNT, PI3K-AKT, NOTCH, Integrin, Hedgehog, Hippo etc. have been shown to be activated by TGF-β in different contexts. The canonical TGF-β/SMAD pathway has been shown to be essential for both tumor suppressive and tumor promoting actions of TGF-β. Although the non-canonical signalling pathways have been shown to be context dependent, the exact mechanisms have not been elucidated. In previous studies, we have shown the importance of non-canonical TGF-β signaling in normal vs. carcinoma cells. However, there has been no study that addressed the differential effects of TGF-β on cells of connective tissue origin. To throw light on such questions we have undertaken this study with the following objectives: 1) Whole genome expression profiling of TGF-β targets in normal fibroblasts, transformed fibroblasts and sarcoma cells 2) Elucidation of non-canonical signaling pathways differentially regulated by TGF-β 3) Identification and characterization of novel TGF-β targets The cell-lines chosen for the study are: 1) hFhTERT (human foreskin fibroblasts immortalized with human terminal telomerase); 2) hFhTERT-LTgRAS (hFhTERT transformed with SV40 large T antigen and activated RAS); and 3) HT1080 (fibrosarcoma). We performed whole genome expression profiling using 4×44K Agilent Human Whole Genome Oligonucleotide Arrays. Analysis of the microarray results revealed that TGF-β regulated a large number of genes in all the three cell-lines but few targets were found to be commonly regulated between any two or all the three cell-lines. 5291 genes were differentially regulated by TGF-β between hFhTERT and hFhTERT-LTgRAS and 2274 genes were differentially regulated by TGF-β between hFhTERT and HT1080 cells. Gene set enrichment analysis (GSEA) of these two gene lists revealed enrichment of similar gene sets in the HT1080 and hFhTERT-LTgRAS cells compared to the hFhTERT cells. MAPK signaling pathway components were enriched in the hFhTERT cells. Closer inspection revealed that several upstream regulators of the MAPK pathway were in fact down-regulated by TGF-β in these cells compared to both hFhTERT-LTgRAS and HT1080 cells suggesting a depression of the MAPK pathway by TGF-β in the hFhTERT cells. Assessment of the phosphorylation status of ERK1/2 and p38 MAPK proteins after TGF-β treatment showed that both ERK1/2 and p38 MAPK pathways were not activated in response to TGF-β in the hFhTERT cells. On the other hand in hFhTERT-LTgRAS and HT1080 cells, both ERK1/2 and p38 MAPK were activated post TGF-β treatment. Activity of the AP1 and SMAD responsive p3TP-lux reporter plasmid was dependent on only the SMAD pathway in hFhTERT cells while in the hFhTERT-LTgRAS and HT1080 cells both MAPK and SMAD pathway were found to regulate the expression of the p3TP-lux reporter. This suggests activation of MAPK and SMAD pathways in transformed and tumor cells while there is no activation of MAPK in normal cells of mesenchymal origin. Components of the WNT signaling pathway such as WNT ligands WNT4, and WNT11, frizzled receptors, FZD4, FZD8 and FZD9, regulators like SFRP1, SFRP2, AXIN2 and several targets of the WNT-β-catenin pathway were regulated by TGF-β in the hFhTERT cells but not in the hFhTERT-LTgRAS and HT1080 cells suggesting a positive regulation of the pathway by TGF-β in the hFhTERT cells. Indeed, TGF-β induced the activity of the WNT responsive reporter, pTOP-FLASH in the hFhTERT cells but not in the hFhTERTLTgRAS and HT1080 cells. WNT4 and WNT11 were two of the novel targets of TGF-β identified in hFhTERT cells. Further experiments suggested that TGF-β conferred regulation of these genes was specific to the fibroblast cells since induction of these genes by TGF-β was not observed in any of the cancer cell lines or in HaCaT cells. Some recent studies have demonstrated remodelling of cytoskeleton in epithelial cells by the non-canonical WNT ligands such as WNT5a, WNT4 and WNT11. WNT4 has also been shown to be required for the maintenance of α-SMA levels in smooth muscle cells. In this study we have shown that WNT4 can induce α-SMA in the hFhTERT cells leading to their activation. TGF-β conferred activation of these cells was also found to be dependent on the presence of WNT4. In brief, our study identified differentially activated pathways by TGF-β in immortal and transformed fibroblasts. WNT4 was identified as a crucial molecule required for the TGF-β conferred activation of fibroblasts.

Spermatogenesis is a complex and coordinated process of formation of sperms from the precursor spermatogonia, occurring inside the unique environment existing in the seminiferous epithelium. This process of development, characterized by concomitant changes in the cellular morphology, metabolism and differential gene expression, can be divided into 3 distinct phases: i) proliferation of the spermatogonia through mitosis; ii) meiosis or reduction division, which commences with transformation of the type B spermatogonia into primary spermatocytes and their subsequent entry into the meiotic prophase I. These primary spermatocytes, divide to form secondary spermatocytes, and then divide again to form haploid round spermatids; (iii) spermiogenesis or differentiation and maturation of the round spermatids without further division to form the unique spermatozoa (Kerr and De Kretser, 2006, Clermont, 1966, Heller and Clermont, 1964). This complex process of division and differentiation is regulated at three distinct levels: i) The extrinsic level where the gonadotropins and testosterone regulate gene expression in the germ cells sustaining their survival and differentiation (French, 2012); ii) The interactive regulation that involves interactions between the somatic cells such as the Sertoli cells and the germ cells; iii) The intrinsic gene expression associated with each step of development of the germ cells (Eddy, 2002) wherein each stage of differentiation is accompanied by precise stage-specific differential gene expression. (Kleene, 1996, Kierszenbaum et al., 2003, Sassone-Corsi, 2002, Kleene, 2001, Sassone-Corsi, 1997). Any alterations in this gene expression pattern leads to disruption and/or arrest of spermatogenesis at various stages, causing male infertility (Zorrilla and Yatsenko, 2013, Krausz et al., 2015). Many studies have been focused on investigating the underlying molecular mechanisms governing the process of germ cell development such as self-renewal, meiotic recombination and differentiation (Hecht, 1998, Grootegoed et al., 2000, Robles et al., 2017). Analysis of differential gene expression in isolated and purified populations of different germ cells have been very useful in the understanding of the genetic regulation of human spermatogenesis by providing information about the cell type-specific gene expression and regulation. (Meistrich et al., 1973, Bellvé, 1993, Meistrich et al., 1981, Chalmel et al., 2007). However, these methods are limited by the large amounts of tissue required, which is difficult to obtain in the case of humans (Schultz et al., 2003). Large-scale gene expression studies and the “omics revolution” have also helped in identifying some of the regulators of spermatogenesis (Carrell et al., 2016). In spite of advances in the current understanding of the regulation of spermatogenesis, the exact molecular mechanisms of how the genetic and epigenetic alterations affect human spermatogenesis are still unclear (Neto et al., 2016). The present study is an attempt to investigate the human testicular gene expression pattern in the germ cells of patients with various types of azoospermia, and correlate the same to infertility. Comparative analysis of the testicular transcriptomes of infertile individuals (with arrested spermatogenesis) with the control, fertile individuals (with normal spermatogenesis) would allow identification of the cell type-specific altered genes. Analysis of these genes would provide an insight into the genetic regulation of the progress of spermatogenesis as well as allow identification of the crucial genes responsible for the arrest. The first step in this study was to ascertain the exact status of spermatogenesis in patients’ testes. Forty-four azoospermic patients were classified clinically into two major groups – obstructive (OA) and non-obstructive (NOA) azoospermia and further classified using flow cytometric analysis of the germ cells. The patients with OA exhibited presence of the diploid, tetraploid and haploid cells indicating complete spermatogenesis (Group I: DTH). The patients with NOA showed incomplete spermatogenesis with arrest at either the meiotic stage showing the presence of diploid and tetraploid cells, but not the haploid cells (Group II: DT), or at the pre-meiotic stage with only diploid cells (Group III: D). This was further verified by RT-PCR analyses for specific markers for different testicular cells. The Group I patients showed expression of markers specific for the Leydig cell (LHCGR, HSD3B2 and HSD17B3), the Sertoli cell (FSHR, KITL), spermatogonia (KIT), tetraploid cells (CCNA1, LDHC) and haploid cells (PRM1). The Group II patients showed expression of CCNA1 and LDHC, but not of PRM1. The Group III patients did not express any of the haploid or tetraploid specific markers. The germ cell pattern was further confirmed by histology where a clear difference was seen across the groups in accordance with their flow cytometric profiles. Subsequent to grouping of the patient samples based on their testicular germ-cell pattern, microarray analysis was carried out with representative samples from each group leading to identification of diploid-/tetraploid-/haploid-specific (D/T/H) genes. The enrichment, probable pathways and network interactions of these identified genes were analyzed and found to be in agreement with the classification made in this study. Further, based on their network interactions, the genes that were under multiple modes of regulation and the transcription factors that regulated multiple pathways were selected for further analysis. In absence of an in-vitro system to study germ cell differentiation, the importance of the selected genes in the progression of human spermatogenesis was analyzed from the data extrapolated from information available in the literature about expression of each gene in the human testes (wherever available), known function of the genes in various somatic cells, function in developing and adult testes of model organisms and the data from the knockout or transgenic animals where disruption of the gene/s resulted in an arrest or disruption of spermatogenesis. Expression of all the putative crucial genes was analyzed in all the patients including the control patients at the transcript level and three selected genes (one from each group- D, T and H) were further validated at the protein level using immunohistochemistry. All the genes showed a similar pattern of amplification in the different groups of patients to the pattern observed from the microarray. The diploid-specific genes (selected based on the available literature) were mainly the inhibitors or regulators of the cell cycle (CDKN1A, GADD45A, FOXM1) (Xiong et al., 1991, Jin et al., 2002, Laoukili et al., 2005) and regulators of cellular proliferation (KLFs, FOS, SRF, ATFs, SMADs) (Garrett-Sinha et al., 1996, Persengiev and Green, 2003, Angel and Karin, 1991, Ten Dijke et al., 2002). Six diploid-specific genes that were potential regulators of spermatogenesis were identified to be probable causes for the arrest of spermatogenesis at the pre-meiotic stage. CDKN1A showed elevated expression at the transcript level which suggested that DNA-damage induced proliferation check (mediated through CDKN1A) in the diploid cells probably prevented these cells from entering meiosis. This was further verified at the protein level by immuno-staining for CDKN1A. Further, GADD45A, KLF4, FOS, MCL1 and SERPINE1 were identified as genes crucial for transition from the diploid to the tetraploid stage and their aberrant expression correlated to the arrest of spermatogenesis in the Group D patients. Six tetraploid-specific genes and four haploid-specific genes were identified to be potential regulators of the tetraploid-haploid transition and responsible for the meiotic arrest. Over expression of the pro-inflammatory genes such as CCL3, IL1B and IL8 (Guazzone et al., 2009) was seen in the testis of the arrested patients which suggested that there was a potential alteration of the normal testicular micro-environment. Expression of EGR2 (a spermatogonial-maintenance gene controlling mitosis (Joseph et al., 1988)) was seen in the nucleus of spermatocytes in group DT patients which indicated its role in the meiotic arrest. To understand the role of the haploid-specific genes in the context of spermatocyte differentiation, only those genes whose expression are reported in the spermatocytes and persist till the spermatid stage were selected. Lack of expression of CST8 was identified to be potentially responsible for loss of germ cell integrity, and the loss of GGN expression in the Group DT patients seemed to be a significant contributor to the genotoxic stress in these patients. In the arrested patients RFX2 (reported to be master regulator of spermiogenesis (Wu et al., 2016)) was seen to be down regulated at the transcript level which indicated its role in the control of meiosis. This was further confirmed by IHC, where expression of RFX2 was seen to be present in the tetraploid cells of the Group DTH patients while no expression was seen in the tetraploid cells of Group DT patients. Thus, this study identified a role for RFX2 in the regulation of meiosis in humans, similar to the findings reported in rats (Horvath et al., 2009). The study also identified autophagy as a mechanism for the clearance of the arrested cells in NOA patients. IHC data using αLC3B showed that autophagy was up regulated in the arrested patients as compared to the Group DTH patients suggesting its role in cell survival and recycling of nutrients. Further, in-situ TUNEL labeling of tissue sections from the different groups (DTH, DT and D) revealed that there were no difference in the status of apoptosis in the azoospermic patients. The latter observation further corroborated with the elevated expressions of CDKN1A, GADD45A, MCL1, TNFAIP3 (reported to ensure cell survival by negatively modulating apoptosis) as seen in the NOA patients. In conclusion, this study identifies several genes that control the progression of spermatogenesis, including the genes whose alterations contribute towards an arrest in spermatogenesis, especially in azoospermia. These identified genes may be used as novel markers in the diagnosis of male infertility. The study opens up the possibility of using the identified genes as future therapeutic targets using small molecular regulators for treatment of infertility as well as targets for male contraception. The study also identifies a novel role for autophagy in patients with NOA which opens up new avenues for further investigation. Thus, this study is the beginning of understanding the complex events that regulate spermatogenesis.

The corpus luteum (CL) is a transient endocrine structure formed from the remnants of an ovulated follicle with the primary purpose of producing progesterone (P4), a hormone vital for the establishment and maintenance of pregnancy. The precise regulation of CL function is essential for normal reproductive cycles and maintenance of early pregnancy. In mammals, the pituitary hormones prolactin (PRL) and luteinizing hormone (LH) function as luteotrophic factors during pregnancy, and these two hormones form a functional luteotrophic complex to control CL function in rodents. The mechanistic underpinnings of the luteotrophic actions of PRL and LH, as well as the interplay between the two hormones are poorly understood, and has been the focus of the current investigation. There are several limitations involved in studying luteal function under cell culture conditions. Hence, in vivo animal models employing dopaminergic receptor agonist, 2-Bromo-α-Ergocryptine Mesylate (CB-154; inhibits pituitary PRL secretion) and GnRH receptor antagonist, cetrorelix (CET; inhibits pituitary LH secretion) have been standardized for purposes of examining the roles of PRL and LH in the regulation of CL structure and function in rats. Administration of CB-154 or CET to pregnant rats caused inhibition of CL function and concomitant loss of conceptuses. The CB-154 treatment induced loss of implants was determined to be the result of inhibition of luteal function, rather than the non-specific effects of CB-154 or requirement of PRL for uterine maintenance of implants. To understand how PRL and LH regulate luteal function, targets of PRL and LH in the rat CL needs to be established; however, this has not been well defined by previous studies. The present study observed that CB-154 induced inhibition of luteal function was gradual in its onset; hence, transcriptional changes of genes involved in steroid genesis were examined. mRNA expression of genes involved in P4 production were found to be down regulated, while 20α-hydroxysteroid dehydrogenate (20α-HSD), a P4 catabolizing enzyme was unregulated by CB-154 treatment. CET treatment also had a similar effect on mRNA expression of steroidogenic genes. Interestingly, mRNA expression of the steroidogenic acute regulatory protein (StAR), a key regulator of steroid genesis was not regulated by CB-154 or CET treatment. The luteolytic factor PGF2α also inhibited CL function in pregnant rats but did not down regulate mRNA expression of StAR. However, examination of phospho-StAR (Ser-195), the activated form of StAR, during CET and PGF2α-induced luteolysis suggested that regulation of StAR in the CL of pregnant rats might primarily be at the level of phosphorylation. PRL has been implicated in maintaining luteal expression of LH/choriogonadotrophin receptor (LH/CGR), the cognate receptor for LH. Hence, the luteotrophic actions of PRL may be indirect, by way of regulating LH signalling. Hence, the importance of the LH/CGR pathway and its regulation were examined. LH/CGR mRNA expression was found to correlate with CL function, with CET and CB-154 treatments resulting in down regulation of LH/CGR mRNA expression. Further, CB-154 treatment down regulated LH/CGR pre-mRNA levels, suggesting a role for PRL in the regulation of LH/CGR transcription. mRNA expression of LRH-1, a constitutively active transcription factor previously reported to be important in CL function was down regulated by both CB-154 and CET treatments and hence correlated with LH/CGR mRNA expression. Further, luciferase assays in HeLA cells transiently expressing LRH-1 suggests its involvement in activating the LH/CGR promoter. Estrogen receptor (ER)-α and ER-β also appear to correlate with LH/CGR expression and may play a role along with LRH-1 in the regulation of LH/CGR mRNA expression in the CL of pregnant rats. To examine mechanisms by which PRL may regulate its downstream targets, pathways employed by PRL in the CL of pregnant rats were analysed. The Akt pathway including downstream targets were down regulated by CB-154 treatment. The pathway was found to be regulated at the level of Akt1 mRNA expression. Hence, actions of PRL may regulate the survival of CL. This study has also made observations of LH playing a similar role in survival of the CL. The results of these studies taken together, shed light on the regulation of CL structure and function by PRL and LH, and provide molecular evidence for the two hormones having similar downstream targets and functioning as a luteotrophic complex in pregnant rats, which could only mean a robust interaction between the signalling pathways employed by the two hormones.

Oral submucous fibrosis (OSF) is a debilitating irreversible fibrotic condition of the oral cavity. It is characterized by inflammation and ultimately results in trismus. Patients face difficulty in speaking, swallowing and chewing due to restricted mouth opening (trismus). This disease is also categorized as an oral premalignant disorder (OPMD). Recent reports cite a conversion rate of 10% from OSF to oral squamous cell carcinoma (OSCC). Epidemiological studies and case reports over the years have correlated the habit of chewing areca nut (Areca catechu) to the manifestation of OSF. It is a major cause of concern in the South and South East Asian parts of the world where areca nut is cultivated and routinely consumed. There are an estimated 700 million areca nut chewers around the globe with 0.5% of the population in the Indian subcontinent being affected by OSF due to this habit. Previous studies have reported differential gene expression profile and up regulation of the pro-fibrotic transforming growth factor-β (TGF-β) pathway in OSF. However, detailed molecular mechanisms for the pathogenesis of this disease are still unclear despite our knowledge about the etiological agent (areca nut) responsible for its progression. Therefore, to gain insights into the etiopathogeneses of OSF, following objectives were undertaken:  To study the gene expression changes induced by areca nut and pro-fibrotic cytokine TGF-β in primary fibroblast cells, and their implications in OSF.  To elucidate the mechanism of TGF-β signal activation in epithelial cells by areca nut. Fibroblast cells are the effectors in all fibrotic disorders. Therefore, it is essential to study the response of this cell type in fibrosis. With prior knowledge of the activation of TGF-β pathway in OSF and the etiological agent of this disease being areca nut; we wanted to study the differential gene response of fibroblasts to these two agents. For this purpose, human primary gingival fibroblasts (hGF) were used as a model system to study the global gene expression profile regulated by areca nut and/or TGF-β. hGF cells were treated with sub-cytotoxic dose of areca nut (5 µg/ml) with and without TGF-β (5 ng/ml) for 72 hours and microarray was performed. The results revealed 4666 genes being differentially regulated by areca nut in hGF cells while TGF-β regulated 1214 genes. Both of them together differentially regulated 5752 genes. 413 genes which were commonly regulated by areca nut and TGF-β were observed to have enhanced regulation with a combined treatment of areca nut, together with TGF-β. This result pointed towards the potential role of both areca nut and TGF-β in modulating fibroblast response. To further assess the role of areca nut in OSF manifestation, we first compared the transcriptome profile induced by it in epithelial cells with fibroblast cells. Areca nut was found to induce differential response in these two cell types which corroborates with the disease pathology wherein; epithelial atrophy is observed and conversely fibroblasts are proliferative. To extend these observations we compared the areca nut induced profile in epithelial cells with OSF differential profile and found that a majority of the genes regulated by areca nut which were common with OSF are regulated by TGF-β. Similarly, areca nut and TGF-β regulated profile in fibroblast cells overlapped significantly with OSF profile. Additionally, areca nut and TGF-β treatment positively enriched matrix associated and metabolic pathways among others which are reported to be differentially regulated in OSF. These observations also highlighted the importance of combined actions of areca nut and TGF-β in OSF manifestation. To test the physiological importance of combined actions of areca nut and TGF-β in the context of OSF; activation of fibroblasts by these treatments was assessed. Treatment of fibroblasts with areca nut and TGF-β enhanced the expression of myofibroblast markers αSMA and γSMA with a concomitant increase in the contractile property when compared to areca nut or TGF-β treatment alone. Further, we observed that areca nut did not regulate any of the TGF-β ligands or receptors in fibroblasts, whereas it induced TGF-β2 in epithelial cells. Therefore, this invoked a possible epithelial-mesenchymal interaction that may exist in OSF pathogenesis. To test this possibility in-vitro, epithelial cells were treated with areca nut and the secretome of these cells was put on hGF cells to study the regulation of fibrosis associated genes. This treatment enhanced the regulation of fibroblast activation markers (αSMA and γSMA) as compared to direct areca nut treatment. This increase in regulation was abrogated when induction of TGF-β2 was compromised in epithelial cells. Similar results were obtained for the regulation of other genes (TGM-2, THBS-1, EDN1, LOXL3, PLOD2, TMEPAI, TGFBI, CTGF, BMP1, LMIK1). Therefore, we concluded that TGF-β which is secreted in response to areca nut by epithelial cells influences fibroblasts in combination with areca nut to enhance fibrosis response. Furthermore, the secretome of untreated epithelial cells was found to down regulate the basal expression of fibrosis related genes in fibroblasts, invoking a role for epithelial secretome in regulating the fibrosis progression. Our data highlighted the importance of TGF-β’s influence on fibroblast response in OSF, but the mechanism for the regulation of this cytokine was not known. Areca nut did not induce TGF-β ligands in fibroblast as discussed above, but previous data from our group had reported areca nut mediated up regulation of TGF-β2 in epithelial cells. Therefore, we further elucidated the mechanistic details for this induction using immortalized keratinocytes (HaCaT and HPL1D) and correlated these in OSF tissues. The kinetics of the induction of TGF-β signaling by areca nut (5 µg/ml) in epithelial cells was established. Areca nut induced TGF-β2 transcript, protein and activated the canonical signaling (pSMAD2/3) at 2 hours post treatment, which persisted till 24 hours. The regulation of TGF-β2 mRNA at 2 hours was dependent on active transcription but was independent of protein translation whereas the activation of signaling (pSMAD2) required both transcription and translation at this time point. This warranted probing for the role of TβR-I in the activation of TGF-β signal by areca nut. A small molecule inhibitor was used to abrogate the kinase activity of TβR-I. Areca nut induced TGF-β2 mRNA at 2 hours even in the presence of TβR-I inhibitor whereas the induction was compromised at 24 hours although the activation of SMAD2 at both 2 and 24 hours was compromised in the presence of TβR-I. This result signified that induction of TGF-β signaling was dependent on the TβR-I activity at early and late time points, but the transcription of the ligand was independent of the receptor activity at early time point. These results indicated the activation of some other pathway by areca nut which could regulate the transcription of TGF-β2 and thereby activate TGF-β signaling in epithelial cells. To explore this possibility, a panel of pathway inhibitors was used and only JNK inhibitor compromised areca nut induced TGF-β2 mRNA and pSMAD2. The results were corroborated by transient knockdown of JNK1 and JNK2. Further, JNK was phosphorylated at 30 minutes to 2 hours by areca nut treatment on epithelial cells. This activation was found to be independent of TβR-I activity. In good correlation, activated JNK1/2 was also detected in OSF tissues and was not detectable in normal tissues. Since JNK activation was found to be a pre-requisite for areca nut induced TGF-β signal activation; we further explored the mechanism of JNK activation by areca nut itself. Areca nut mediated activation of JNK was found to be dependent on muscarinic acid receptor, Ca2+/CAMKII and ROS. Inhibition of these significantly compromised areca nut induced pJNK. In line with this, inhibition of muscarinic acid receptor activity, CAMKII and ROS also abrogated areca nut mediated induction of TGF-β2 mRNA and pSMAD2. The regulation of TGF-β signaling by areca nut in epithelial cells was dependent on transcription, and JNK activity was essential for this. We further sought to explore transcription factors which were regulated by JNK and therefore could possibly induce TGF-β2 promoter activity. ATF2 and c-Jun transcription factors were found to be induced at 30 minutes by areca nut and this up regulation also persisted till 24 hours. Further, activation of both ATF2 and c-Jun was dependent on JNK but independent of TβR-I activity. Moreover, areca nut treatment induced translocation of these phoshorylated transcription factors in the nucleus of epithelial cells. Additionally, pATF2 and p-c-Jun were enriched on TGF-β2 promoter after 2 hours of treatment by areca nut. To investigate the importance of this enrichment and regulation of TGF-β signal activation by areca nut, we transiently knocked down these proteins and studied the regulation of TGF-β2. Areca nut induced TGF-β2 mRNA and pSMAD2 was abrogated upon ATF2 and c-Jun knockdown, implicating JNK mediated activation of ATF2 and c-Jun in areca nut induced TGF- β signaling. To explore the significance of this mechanism in OSF, immunohistochemical staining for pATF2 and p-c-Jun was performed on OSF and normal tissues. Both the transcription factors were found in the nuclei of OSF tissues whereas their expression was not detected in normal tissues. This expression also correlated with the previously reported activation of SMAD2 in OSF tissues by our group. Hence, ATF2 and c-Jun were observed to be important in areca nut mediated TGF-β signaling in OSF. In conclusion, the work described in this thesis provides mechanistic details into OSF etiopathogenesis. Combined actions of areca nut and TGF-β induced a response in fibroblasts akin to OSF. Our results advocate a role for epithelial secreted factors in influencing fibroblast response in both normal and disease (OSF) conditions. Further, importance of TGF-β in OSF has been elucidated in terms of enhancing the fibroblast response to areca nut. We have also elucidated the mechanism for areca nut mediated activation of TGF-β signaling and have identified the contribution of JNK/ATF2/Jun axis in this process. This work can impact the management of oral submucous fibrosis by providing newer targets for treatment.

Insulin like Growth Factor Binding Proteins (IGFBPs) 1 to 6 have important physiological functions of regulating half life and bioavailability of Insulin like Growth Factors (IGFs). Consequently, these have been known to play important roles in embryonic development, postnatal growth and disease conditions like cancer. However, the physiological roles of IGFBPs are diverse and not restricted only to the IGF regulation. These molecules are found to be tumor suppressors or promoters depending on the physiological contexts. IGFBP-2 has been established as a tumor promoter and found to be unregulated in several cancers including breast, ovarian, prostate cancer and glioblastoma (GBM). Various in vitro and in vivo studies have convincingly demonstrated the role of IGFBP-2 in inducing tumor cell proliferation, migration, invasion and chemoresistance. Increased plasma and tissue levels of IGFBP-2 have been associated with poor clinical outcome with respect to patients’ response to the therapy, relapse and overall survival. Various studies so far have demonstrated the role of IGFBP-2 in promoting glioma cell proliferation, migration, invasion, chemoresistance and determining stamens of GICs (Glioma Initiating Cells). However, the exact mechanisms underlying these functions remain unknown. Apart from being a diagnostic and prognostic indicator, IGFBP-2 has also been proposed as a therapeutic target. Therefore it is essential to understand mechanistic insights into pro-tumorigenic functions of IGFBP-2. Apart from the conventional function of regulating IGFs, IGFBP-2 has been shown to have several IGF independent functions. In a previous study, we reported IGFBP-2 as an upstream regulator of β-catenin signaling pathway in breast cancer. Interestingly, this study linked the association of higher expression of IGFBP-2 and β-catenin with the lymph node metastasis status of breast cancer. β-catenin signaling has been considered as one of the most important pro-tumorigenic pathways in several cancers including glioblastoma. Considering the importance of IGFBP-2 and β-catenin signaling pathways in glioblastoma, it becomes important to evaluate regulation of β-catenin activity by IGFBP-2 in glioma and address its clinical relevance. With this aim, the objectives of this study are,  To study mechanism of IGFBP-2 mediated regulation of β-catenin signaling in glioma cells and prognostic significance of IGFBP-2 and β-catenin expression in GBM tissues.  Isolation of human single chain variable fragment (scFv) against IGFBP-2 and its characterization as an inhibitor for IGFBP-2 pro-tumorigenic functions. Towards this, we established stable IGFBP-2 knockdown U251 cell line and IGFBP-2 over expressing LN229 and U87 cell lines. IGFBP-2 modulation in these glioma cell lines did not alter the rate of proliferation but there was a significant effect on cellular migration and invasion. In case of U251 cell line, there was a significant decrease in the intracellular levels of β-catenin while in IGFBP-2 over expressing cell lines there was a marked increase in intracellular β-catenin suggesting that IGFBP-2 is involved in the regulation of β-catenin in these cells. It was observed that this regulation of β-catenin was not because of its transcriptional regulation or regulation of canonical Wnt ligands Wnt1, Wnt2 and Wnt3a. To further delineate the pathway and understand the mechanism behind regulation of β-catenin, upstream regulators of β-catenin were analyzed. GSK3β is an important negative regulator of β-catenin which primes it for ubiquitination and proteasomal degradation. Phosphorylation of GSK3β at Ser9 position renders this enzyme inactive. In our study, it was observed that there was a significant downregulation of p-GSK3β in U251 cells with IGFBP-2 knockdown and upregulation in IGFBP-2 over expressing cell lines. Overexpression of IGFBP-2 in LN229 and U87 cell lines resulted in considerable decrease in the GSK3β mediated phosphorylation of β-catenin. This study unequivocally established that regulation of β-catenin by IGFBP-2 is via inactivation of GSK3β. Furthermore, regulation of GSK3β was found to be due to action of FAK following binding of IGFBP-2 to integrins. The expression pattern of IGFBP-2 and β-catenin protein in the tumor tissues of 112 GBM patients was studied and its correlation with patient survival was analysed. In this analysis it was observed that co-expression of IGFBP-2 and β-catenin is a strong predictor of patient prognosis. These results further implied the importance of understanding IGFBP-2 and β-catenin association in GBM pathology. One of the interesting observations in our study is that, not only full length IGFBP-2 protein but also C-terminal domain of IGFBP-2 was sufficient to regulate β-catenin and other IGFBP-2 mediated functions. This strongly asserts the importance of C-terminal region of IGFBP-2 as a tumor promoter. Towards an attempt to develop an inhibitor for IGFBP-2 actions, we screened a human single chain variable fragment (scFv) library using phage display technique. From this screening, one scFv (B7J) was identified which was a binder of full length IGFBP-2 as well as C-terminal domain of IGFBP-2. This scFv showed inhibition of IGFBP-2-cell surface interaction and also efficiently inhibited IGFBP-2-induced signaling pathways like ERK, FAK and GSK3β/β-catenin. B7J treatment also neutralized regulation of IGFBP-2 transcriptional targets like MMP2 and CD24. Gelatin zymography indicated the ability of B7J to decrease matrix metalloprotease activity in the conditioned medium of glioma cells. These effects ultimately reflected on the IGFBP-2-induced cellular migratory and invasive behaviour which was largely abrogated by B7J scFv treatment. Considering the therapeutic importance of scFvs because of their small size, better tumor penetration and tumor retention capacity than full length antibody molecules, such kind of strategy could be of great importance in the management of GBM. Altogether, this study provides a mechanistic insight of IGFBP-2 mediated actions involving integrin/FAK/GSK3β/β-catenin pathways and the possible role of this crosstalk in the aggressiveness of glioblastoma. This study also provides a proof of principle that an inhibitor like anti IGFBP-2 scFv could be of importance for controlling invasive glioblastoma.

Cancer progression is a multi-step process requiring cells to acquire specific properties that aid the neoplastic growth. One such property is the ability to survive in the absence of matrix-attachment, a critical necessity for cells to traverse in circulation and seed metastases. Therefore, understanding the signalling mechanisms that protect cells from undergoing death in matrix-deprived condition, termed as anoikis, is important. We have used two systems to study this, one involving experimental transformation model, and another involving cancer cell lines. In the in vitro transformation model system involving the serial introduction of oncogenes, the ability to survive in anchorage-independent condition and generate spheres/colonies was dependent on the presence of the Simian Virus Small T antigen, SV40 ST. We identified that the viral antigen mediates its effects, at least in part, by activating the master metabolic regulator and cellular stress kinase AMP-activated protein kinase (AMPK) leading to maintenance of energy homeostasis. Consistent with this, our lab has previously identified both activation of AMPK upon matrix-deprivation in breast cells, as well as its requirement for survival under these conditions. However, a pathway often associated with survival under matrix-deprivation is the PI3K/Akt pathway. Surprisingly, we observed an AMPK-dependent decrease in Akt activity under conditions of matrix-detachment. Since this was contrary to the general notion, we probed deeper into a possible crosstalk between these two kinases. Our work revealed that AMPK activation in suspension inhibits Akt via upregulation of a known Akt phosphatase, pleckstrin homology domain leucinrich repeat protein phosphatise (PHLPP). We further show that the AMPK-PHLPP-Akt signalling axis is important for anoikis-resistance and metastasis. In addition, our results point to a yet unidentified protumorigenic role of PHLPP in breast cancer progression. With an aim to identify cellular proteins differentially regulated upon AMPK activation in breast cancer cells, we undertook a proteomics approach. Using 2-dimensional gel electrophoresis followed by mass spectrometric analysis, we identified some candidate proteins. We have validated the increase in levels of one of these proteins, annexin A2, in cancer cells upon AMPK activation. In summary, the present study unveils novel oncogenic functions of AMPK in cancer cells under the stress of matrix-deprivation. Furthermore, our results elucidate a double-negative feedback loop between two critical cellular kinases AMPK and Akt, and also identify a novel pro-tumorigenic role of PHLPP in breast cancer. In addition, we identify PHLPP and annexin A2 as novel proteins upregulated by AMPK in cancer cells. Thus, our results begin to identify pathways utilised by cancer cells to aid anchorage-independent growth, a critical step for cancer metastasis. Based on our results, inhibition of AMPK or perturbation of signalling axes involving AMPK, and PHLPP or annexin A2 might be considered as novel therapeutic approaches to combat cancer progression