Vijayalaxmi Gupta

Role of FYN Kinase in Spermatogenesis: Defects Characteristic of Fyn-Null Sperm in Mice

ABSTRACT FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.

Synthesis and Evaluation of Eight- and Four-Membered Iminosugar Analogues as Inhibitors of Testicular Ceramide-Specific Glucosyltransferase, Testicular β-Glucosidase 2, and Other Glycosidases

Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve ring-closing metathesis and Sharpless asymmetric dihydroxylation and for the four-membered analogues Sharpless epoxidation, epoxide ring-opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6) was moderately active against rat-derived ceramide-specific glucosyltransferase, and four of the other eight-membered analogues were weakly active against rat-derived β-glucosidase 2. Among the four-membered analogues, ((2R,3S,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25) displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of β-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived β-glucosidase, and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC(50) of 600 nM and β-glucosidase (almond) with an IC(50) of 20 μM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied.

Human pancreatic cancer progression: an anarchy among CCN-siblings

Decades of basic and translational studies have identified the mechanisms by which pancreatic cancer cells use molecular pathways to hijack the normal homeostasis of the pancreas, promoting pancreatic cancer initiation, progression, and metastasis, as well as drug resistance. These molecular pathways were explored to develop targeted therapies to prevent or cure this fatal disease. Regrettably, the studies found that majority of the molecular events that dictate carcinogenic growth in the pancreas are non-actionable (potential non-responder groups of targeted therapy). In this review we discuss exciting discoveries on CCN-siblings that reveal how CCN-family members contribute to the different aspects of the development of pancreatic cancer with special emphasis on therapy.

MicroRNA Expression Signatures During Malignant Progression From Barrett's Esophagus

The rapid increase and poor survival of esophageal adenocarcinoma (EAC) have led to significant efforts to promote early detection. Given that the premalignant lesion of Barretts esophagus (BE) is the major known risk factor for EAC, multiple investigators have studied biomarker signatures that can predict malignant progression of BE to EAC. MicroRNAs, a novel class of gene regulators, are small non‐coding RNAs and have been associated with carcinogenesis. MicroRNAs are ideal biomarkers because of their remarkable stability in fixed tissues, a common method for collection of clinical specimens, and in blood either within exosomes or as microRNA‐protein complexes. Multiple studies show potential of microRNAs as tissue and blood biomarkers for diagnosis and prognosis of EAC but the results need confirmation in prospective studies. Although head‐to‐head comparisons are lacking, microRNA panels require less genes than messenger RNA panels for diagnosis of EAC in BE. MicroRNA diagnostic panels will need to be compared for accuracy against global measures of genome instability that were recently shown to be good predictors of progression but require sophisticated analytic techniques. Early studies on blood microRNA panels are promising but have found microRNA markers to be inconsistent among studies. MicroRNA expression in blood is different between various microRNA sub‐compartments such as exosomes and microRNA‐protein complexes and could affect blood microRNA measurements. Further standardization is needed to yield consistent results. We have summarized the current understanding of the tissue and blood microRNA signatures that may predict the development and progression of EAC. J. Cell. Biochem. 117: 1288–1295, 2016.

Structure-Activity Studies of N-Butyl-1-deoxynojirimycin (NB-DNJ) Analogues: Discovery of Potent and Selective Aminocyclopentitol Inhibitors of GBA1 and GBA2.

Analogues of N‐butyl‐1‐deoxynojirimycin (NB‐DNJ) were prepared and assayed for inhibition of ceramide‐specific glucosyltransferase (CGT), non‐lysosomal β‐glucosidase 2 (GBA2) and the lysosomal β‐glucosidase 1 (GBA1). Compounds 5 a–6 f, which carry sterically demanding nitrogen substituents, and compound 13, devoid of the C3 and C5 hydroxy groups present in DNJ/NB‐DGJ (N‐butyldeoxygalactojirimycin) showed no inhibitory activity for CGT or GBA2. Inversion of stereochemistry at C4 of N‐(n‐butyl)‐ and N‐(n‐nonyl)‐DGJ (compounds 24) also led to a loss of activity in these assays. The aminocyclopentitols N‐(n‐butyl)‐ (35 a), N‐(n‐nonyl)‐4‐amino‐5‐(hydroxymethyl)cyclopentane‐ (35 b), and N‐(1‐(pentyloxy)methyl)adamantan‐1‐yl)‐1,2,3‐triol (35 f), were found to be selective inhibitors of GBA1 and GBA2 that did not inhibit CGT (>1 mm), with the exception of 35 f, which inhibited CGT with an IC50 value of 1 mm. The N‐butyl analogue 35 a was 100‐fold selective for inhibiting GBA1 over GBA2 (Ki values of 32 nm and 3.3 μm for GBA1 and GBA2, respectively). The N‐nonyl analogue 35 b displayed a Ki value of ≪14 nm for GBA1 inhibition and a Ki of 43 nm for GBA2. The N‐(1‐(pentyloxy)methyl)adamantan‐1‐yl) derivative 35 f had Ki values of ≈16 and 14 nm for GBA1 and GBA2, respectively. The related N‐bis‐substituted aminocyclopentitols were found to be significantly less potent inhibitors than their mono‐substituted analogues. The aminocyclopentitol scaffold should hold promise for further inhibitor development.

A Tissue Retrieval and Postharvest Processing Regimen for Rodent Reproductive Tissues Compatible with Long-Term Storage on the International Space Station and Postflight Biospecimen Sharing Program

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at −80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASAs Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Abstract 84: CCN1/Cyr61 regulation of gemcitabine-resistant phenotype in pancreatic cancer: involvement of CTGF and dCK

With an estimated half a million new cases and similar mortality rates for 2016, pancreatic ductal adenocarcinoma (PDAC) remains a life-threatening and challenging disease to diagnose and treat. As per American Cancer Society (ACS), varying efficacy in different patients has led to an increase in the mortality rate of PDAC. Gemcitabine (GEM) remains the drug of choice either alone or in combination, but is unsuccessful in reducing or curing PDAC in most patients. The limited efficacies of these drugs are due to the acquisition of chemo-resistant characteristics of PDAC. Although several molecular and physiological factors have been shown to correlate with the GEM-resistance, defined molecular mechanism(s) of GEM-resistance remains a mystery. Previous studies have shown that CCN1, which is overexpressed in PDAC and known to associate with PDAC progression, is critical for drug resistance. Here, we found that while the pancreatic cancer cell lines (i.e., Panc-1 and AsPC-1) in which CCN1 is overexpressed are typically GEM-resistant, the knocking down of CCN1 makes them sensitive to GEM. Mechanistic studies revealed that CCN1 regulates two important genes that are directly involved in regulation GEM-sensitivity in PDAC. These include cancer cell-secreted connective tissue growth factor (CTGF), a regulator of desmoplasia, and Deoxycytidine kinase (dCK), an enzyme that enhances gemcitabine sensitivity and efficacy in cancer cells. The deletion of CCN1 in PDAC cells blocks CTGF expression and under co-cultured conditions prevent the growth of alpha-smooth muscle actin (α-SMA)-positive stellate cells, which are required for desmoplastic growth in pancreatic cancers. CCN1 ablation upregulates dCK expression in PDAC cell lines as compared to CCN1 expressed PDAC cells. These two events enhance the anti-proliferative effect of GEM and can be rescued by CTGF-treatment or blocking dCK. In conclusion, CCN1 promotes GEM-resistance in PDAC cell through the regulation of CTGF and dCK and the mechanistic insights provided by these studies may help in designing future therapeutic strategies to combat PDAC. Citation Format: Vijayalaxmi G. Gupta, Gargi Maity, Inamul Haque, Sushanta K. Banerjee, Snigdha Banerjee. CCN1/Cyr61 regulation of gemcitabine-resistant phenotype in pancreatic cancer: involvement of CTGF and dCK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 84. doi:10.1158/1538-7445.AM2017-84

Abstract 4915: Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells

Background and Objective: The epidemiological and experimental studies suggest that alcohol consumption is associated with increased risk for breast cancer development and metastasis. However, the mechanisms of alcohol-induced breast cancer progression and metastasis remain unknown. In this study, we examined the roles of CCN5, a tumor suppressor gene in alcohol induced breast cancer cell migration/invasion, epithelial to mesenchymal transition (EMT) and stemness. Methods: Estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 and immortalized normal human epithelial breast cell line MCF-12A were treated with ethanol at various concentrations in the presence and absence of human recombinant human CCN5 protein (hrCCN5) . Invasive/migratory ability of treated and untreated cells was measured by Boyden chamber assays. The mRNA and protein expression level of CCN5 was determined by real-time qRT-PCR and Western blot. EMT and stemness markers were evaluated by Western blot. Single-cell suspensions from pre-treated cells were re-suspended at a density of 500 cells/ml mammocult media in ultralow attachment dishes. Number as well as the size of the mammosphere in specified experimental set-up was monitored and recorded alternate day for 8-10 days. Results: The studies demonstrated that alcohol promotes significantly the invasive/migratory ability and EMT phenotypes of breast cancer cells through the suppression of CCN5. Interestingly, hrCCN5 protein treatment suppresses the effect of alcohol and reprograms MET (mesenchymal to epithelial transition). In addition, alcohol-induced mammosphere formation efficiency is also suppressed by hrCCN5 protein-treatment. Conclusions: Our findings suggest that the CCN5 signaling plays a preventive role in alcohol-induced reprogramming of tumor plasticity in breast cancer cells. Citation Format: Inamul Haque, Arvind Subramanian, Vijayalaxmi Gupta, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4915. doi:10.1158/1538-7445.AM2017-4915