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Currently, clinicians rely on clinical nomograms to stratify progression risk at the time of diagnosis in patients with prostate cancer (CaP). However, these tools may not accurately distinguish aggressive potential in low-grade CaP. The current study determined the diagnostic potential of 3 molecular markers (ROCK1, RUNX3, and miR-301a) in terms of their ability to identify which low-grade tumors are likely to progress. Real-time PCR and immunohistochemical analysis were used to assess ROCK1, RUNX3, and miR-301a expression profiles in 118 serum and needle biopsy specimens. Expressions of ROCK1 and miR-301a were found to be significantly higher in Gleason 6 and 7 CaP as compared to BPH, while an inverse trend was observed with RUNX3. Further, incorporation of all 3 molecular markers significantly improved clinical nomograms' diagnostic accuracy and correlated with disease progression. Hence, in conclusion, the inclusion of these 3 molecular markers identified aggressive phenotype and predicted disease progression in low-grade CaP tumors at the time of diagnosis.

Notch1 activation triggers significant oncogenic signaling that manifests as enhanced metastatic potential and tumorigenesis in colorectal cancer. Novel small-molecule inhibitors, mainly plant-derived analogs, have low toxicity profiles and higher bioavailability. In this study, we have developed a small molecule, ASR490, by modifying structure of naturally occurring compound Withaferin A. ASR490 showed a growth-inhibitory potential by downregulating Notch1 signaling in HCT116 and SW620 cell lines. Docking studies and thermal shift assays confirmed that ASR490 binds to Notch1, whereas no changes in Notch2 and Notch3 expression were seen in colorectal cancer cells. Notch1 governs epithelial-to-mesenchymal transition signaling and is responsible for metastasis, which was abolished by ASR490 treatment. To further confirm the therapeutic potential of ASR490, we stably overexpressed Notch1 in HCT-116 cells and determined its inhibitory potential in transfected colorectal cancer (Notch1/HCT116) cells. ASR490 effectively prevented cell growth in both the vector (P = 0.005) and Notch1 (P = 0.05) transfectants. The downregulation of Notch1 signaling was evident, which corresponded with downregulation of mesenchymal markers, including N-cadherin and β-catenin and induction of E-cadherin in HCT-116 transfectants. Intraperitoneal administration of a 1% MTD dose of ASR490 (5 mg/kg) effectively suppressed the tumor growth in control (pCMV/HCT116) and Notch1/HCT116 in xenotransplanted mice. In addition, downregulation of Notch1 and survival signaling in ASR-treated tumors confirmed the in vitro results. In conclusion, ASR490 appears to be a potent agent that can inhibit Notch1 signaling in colorectal cancer.

Due to a lack of mechanistic insights, muscle-invasive bladder cancer (MIBC) remains incurable and is one of the most lethal types of cancer in the United States. The present study investigated changes in the molecular signatures of MIBC cells (TCCSUP and HT1376) after treatment with a novel small molecule, ASR488, to gain knowledge of the mechanisms that inhibited MIBC cell growth. ASR488 treatment initiated apoptotic signaling in MIBC cells. Pathway enrichment analysis was used to analyze the changes in function of differentially expressed genes. Gene Ontology analysis, as well as Kyoto Encyclopedia of Genes and Genomes analysis, was also performed. These analyses along with reactome pathway enrichment analyses indicated that the genes upregulated in the ASR488-treated cells are involved in focal adhesion, neurotrophin signaling, p53 signaling, endoplasmic reticulum functioning in terms of protein processing, and pathways related to bladder cancer. The genes downregulated in ASR488-treated MIBC cells were mainly involved in DNA replication, mismatch repair, RNA degradation, nucleotide excision repair and TGFβ signaling (P

The aim of the present study was to investigate the genetic signatures of cadmium-transformed prostate epithelial (CTPE) cells and to identify the potential molecular signaling involved in their malignant transformation. The dataset contained normal prostate epithelial (RWPE-1) and CTPE cells. To further examine the biological functions of the identified differentially expressed genes (DEGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway enrichment analyses were performed. In total, 2,357 DEGs were identified, including 1,083 upregulated genes and 1,274 downregulated genes. GO, KEGG, and Reactome pathway enrichment analyses indicated that upregulated genes were significantly enriched in ECM–receptor, focal adhesion, TGFβ signaling, and syndecan interactions, while downregulated genes were mainly involved in cell cycle regulation, arachidonic acid metabolism, oxidative phosphorylation, and folate biosynthesis (p < 0.05). The top upregulated (SATB1 (p

Automated long-term mapping and climate niche modeling are important for developing adaptation and management strategies for rubber plantations (RP). Landsat imageries at the defoliation and refoliation stages were employed for RP mapping in the Indian state of Tripura. A decision tree classifier was applied to Landsat image-derived vegetation indices (Normalized Difference Vegetation Index and Difference Vegetation Index) for mapping RPs at two-three years intervals from 1990 to 2017. A comparison with actual plantation data indicated more than 91% mapping accuracy, with most RPs able to be identified within six years of plantation, while several patches were detected after six years of plantations. The RP patches identified in 1990 and before 2000 were used for training the Maxent species distribution model, wherein bioclimatic variables for 1960–1990 and 1970–2000 were used as predictor variables, respectively. The model-estimated suitability maps were validated using the successive plantation sites. Moreover, the RPs identified before 2017 and the Shared Socioeconomic Pathways (SSP) climate projections (SSP126 and SSP245) were used to predict the habitat suitability for 2041–2060. The past climatic changes (decrease in temperature and a minor reduction in precipitation) and identified RP patches indicated an eastward expansion in the Indian state of Tripura. The projected increase in temperature and a minor reduction in the driest quarter precipitation will contribute to more energy and sufficient water availability, which may facilitate the further eastward expansion of RPs. Systematic multi-temporal stand age mapping would help to identify less productive RP patches, and accurate monitoring could help to develop improved management practices. In addition, the existing RP patches, their expansion, and the projected habitat suitability maps could benefit resource managers in adapting climate change measures and better landscape management.

Background: Evidence from the epidemiological, laboratory and clinical studies suggest that chronic exposure to cadmium (Cd) leads to the development and progression of prostate cancer (CaP). Previously, we reported defective autophagy due to upregulation of Placenta Specific 8 (Plac8) (a phagosome and lysosome fusion regulator) is responsible for Cd-induced transformation of normal prostate epithelial cells. This study mechanistically dissects the upstream signaling that regulates Plac8 function, which drives defective autophagy during Cd-induced transformation. Methods: To examine the molecular interaction between NFκB and Plac8, we developed NFκB/Plac8 overexpression and knockdown in normal prostate epithelial cells (RWPE-1) and cadmium transformed prostate epithelial cells lines (CTPE). In addition, we performed phenotypic, and molecular analysis including promoter-based studies, western blot, and in vivo analysis to determine the interaction between Plac8 and p65 in Cd-exposed prostate epithelial cells. Results: Our results confirmed that overexpression of Plac8 in normal prostate epithelial cells mimics Cd-transformed prostate cells (CTPE) and induces defective autophagy by regulating autophagy-related genes (ATGs) and an enhanced autophagic flux. Conversely, either NFκB or Plac8 knockdown in CTPE cells abrogates autophagy signaling. Further, Cd-exposure failed to induce defective autophagy signaling in transformed cells. Silencing p65 either by genetically or pharmacological inhibitors downregulated Plac8 expression and its survival function in CTPE cells. Hence, we investigated the interaction between NFκB and Plac8 by promoter-based analysis. We found NFκB binding site in the Plac8 promoter, and mutating NFκB binding sites in the Plac8 promoter showed a decline in luciferase activity confirming our hypothesis that NFκB activation is necessary for Plac8 activation and autophagy signaling in CTPE cells. Finally, we confirmed that silencing Plac8 significantly inhibited the tumor growth of CTPE cells compared to vehicle control. Our ongoing studies on xenotransplanted stably knockdown of p65 in CTPE cells may confirm that NFκB activation is responsible for Plac8 function and tumor growth in mice models. Conclusion: In conclusion, our results suggest that NFκB transcriptionally regulates Plac8 function, which drives pro-survival autophagy signaling in Cd-transformed prostate epithelial cells. Citation Format: Vaibhav Shukla, Ashish Tyagi, Venkatesh Kolluru, Uttara Saran, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Activation of NFKB is responsible for defective autophagy in cadmium-induced transformation of prostate cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2999.

As the two largest landlocked countries, Kazakhstan and Mongolia have similar biophysical conditions and socioeconomic roots in the former Soviet Union. Our objective is to investigate the direction, extent, and spatial variation of land cover change at three administrative levels over three decades (1990–2020). We selected three provinces from each country (Aktobe, Akmola, and Almaty province in Kazakhstan, and Arkhangai, Tov, and Dornod in Mongolia) to classify the land cover into forest, grassland, cropland, barren, and water. Altogether, 6964 Landsat images were used in pixel-based classification method with random forest model for image processing. Six thousand training data points (300 training points × 5 classes × 4 periods) for each province were collected for classification and change detection. Land cover changes at decadal and over the entire study period for five land cover classes were quantified at the country, provincial, and county level. High classification accuracy indicates localized land cover classification have an edge over the latest global land cover product and reveal fine differences in landscape composition. The vast steppe landscapes in these two countries are dominated by grasslands of 91.5% for Dornod in Mongolia and 74.7% for Aktobe in Kazakhstan during the 30-year study period. The most common land cover conversion was grassland to cropland. The cyclic land cover conversions between grassland and cropland reflect the impacts of the Soviet Union’s largest reclamation campaign of the 20th century in Kazakhstan and the Atar-3 agriculture re-development in Mongolia. Kazakhstan experienced a higher rate of land cover change over a larger extent of land area than Mongolia. The spatial distribution of land use intensity indicates that land use hotspots are largely influenced by policy and its shifts. Future research based on these large-scale land use and land cover changes should be focused the corresponding ecosystem and society functions.

Meteorological data retrieval is the fundamental process for any hydrological research. Precipitation data collection from some constrained territories like high slant geography and inaccessible areas is exceptionally troublesome. Setting the rain gauges is a matter of expense and timely maintenance. To overcome these issues, satellite sensors producing high spatial and temporal resolution datasets can be utilized in the studies involving precipitation component. These satellite products are affected by biases, and hence, there is a need for calibration and verification by using ground observation data based on the statistical coefficients. In this study, the most accessible satellite data products, i.e., CHIRPS, PERSIANN-CDR and TRMM, are employed to check the accuracies against IMD gridded data for the years 2000–2012 using a statistical approach. Selecting the data product having a high coefficient of correlation and low PBIAS is utmost necessary. The current study was performed based on catchment-to-catchment (C-C) method by comparing IMD gridded data with satellite datasets obtained from Google Earth Engine. The results can highlight the data product which can conquer the issue of data inaccessibility in the investigation territory and can be utilized as reference precipitation dataset for different hydrological applications.

The dynamics of land use/land cover can be studied by using digital change detection techniques which are highly significant for the evaluation and development of management strategies in a region. The environmental and hydrological processes prevailing in the area can be interpreted only by analyzing the alterations in the past and present land use and land cover classes. In view of this, the present study is executed to analyze the typical land use change in the coastal region over the three decades by analyzing historical and current land use/land cover (LU/LC) datasets. Landsat 5 and Landsat 8 satellite datasets were considered for change detection analysis using unsupervised classification method. K-means algorithm, a widely used unsupervised classification technique, was adopted in this study to classify coastal region of Karnataka for the years 1990 and 2019. The level-II classification was performed on LU/LC raster datasets (Landsat 5 and 8) which segregated the entire study area into ten classes, namely agricultural land, barren land, built-up area, water, forest, fallow or cultivated land, scrub forest, sandy area, swampy forest and wetlands. This study encapsulated that about 40% of the study area was occupied by water body followed by forestry with a percentage of around 30%. Major changes were observed in the barren land and scrub forest between 1990 and 2019, where the barren land was replaced by scrub forest in 2019. The accuracy assessment is performed to analyze the efficiency of the algorithm and the precision of the classified image which showed an accuracy of 81% in 1990 and 84% in 2019 demonstrating the ability of an algorithm to classify reliably.

Epidemiological evidence suggests that cadmium (Cd) is one of the causative factors of prostate cancer, but the effect of Cd on benign prostatic hyperplasia (BPH) remains unclear. This study aimed to determine whether Cd exposure could malignantly transform BPH1 cells and, if so, to dissect the mechanism of action. We deciphered the molecular signaling responsible for BPH1 transformation via RNA-sequencing and determined that Cd induced the expression of zinc finger of the cerebellum 2 (ZIC2) in BPH1 cells. We noted Cd exposure increased ZIC2 expression in the Cd-transformed BPH1 cells that in turn promoted anchorage-independent spheroids and increased expression of stem cell drivers, indicating their role in stem cell renewal. Subsequent silencing of ZIC2 expression in transformed cells inhibited spheroid formation, stem cell marker expression, and tumor growth in nude mice. At the molecular level, ZIC2 interacts with the glioma-associated oncogene family (GLI) zinc finger 1 (GLI1), which activates prosurvival factors (nuclear factor NFκB, B-cell lymphoma-2 (Bcl2), as well as an X-linked inhibitor of apoptosis protein (XIAP)) signaling in Cd-exposed BPH1 cells. Conversely, overexpression of ZIC2 in BPH1 cells caused spheroid formation confirming the oncogenic function of ZIC2. ZIC2 activation and GLI1 signaling induction by Cd exposure in primary BPH cells confirmed the clinical significance of this oncogenic function. Finally, human BPH specimens had increased ZIC2 versus adjacent healthy tissues. Thus, we report direct evidence that Cd exposure induces malignant transformation of BPH via activation of ZIC2 and GLI1 signaling.

Chemopreventive effects and associated mechanisms of withaferin A (WA) against intestinal and colon carcinogenesis remain unknown. We investigated the chemopreventive effect of WA on transgenic adenomatous polyposis coli (APC(Min/+)) mouse and chemically induced azoxymethane/dextran sodium sulfate (AOM/DSS) models of intestinal and colon carcinogenesis. Oral WA administration (4 and 3 mg/kg) inhibited tumor initiation and progression of intestinal polyps formation in APC(Min/+) mice and colon carcinogenesis in the AOM/DSS mouse model. WA-administered mice showed a significant reduction in both number [duodenum, 33% (P > 0.05); jejunum, 32% (P < 0.025); ileum, 43% ( P < 0.001); and colon 59% (P < 0.01] and size of polyps in APC(Min/+) mice compared with the respective controls. Similarly, tumor multiplicity was significantly reduced (P < 0.05) in the colon of WA-administered AOM/DSS mice. Pathological analysis showed reduced adenomas and tissue inflammation in WA-administered mouse models. Molecular studies suggested that WA inhibited the expression of inflammatory (interluekin-6, tumor necrosis factor-alpha and cyclooxygenase-2), pro-survival (pAKT, Notch1 and NF-κB) markers in APC(Min/+) and AOM/DSS models. The results suggest that WA is a potent agent for preventing colon carcinogenesis and further investigation is required to show clinical utility of the agent.

Androgen ablation therapy is the primary therapeutic option for locally advanced and metastatic castration-resistant prostate cancer (CRPC). We investigated therapeutic effect of a dietary metabolite Urolithin A (UroA) and dissected the molecular mechanism in CRPC cells. Treatment with UroA inhibited cell proliferation in both androgen receptor-positive (AR+ ) (C4-2B) and androgen receptor-negative (AR- ) (PC-3) cells however, AR+ CaP cells were more sensitive to UroA treatment as compared with AR- CaP cells. Inhibition of the AR signaling was responsible for the UroA effect on AR+ CaP cells. Ectopic expression of AR in PC-3 cells sensitized them to UroA treatment as compared to the vector-expresseing PC-3 cells, which suggests that AR could be a target of UroA. Similarly, in enzalutamide-resistant C4-2B cells, a downregulation of AR expression also suppressed cell proliferation which was observed with the UroA treatment. Oral administration of UroA significantly suppressed the growth of C4-2B xenografts (P = 0.05) compared with PC-3 xenografts (P = 0.069) without causing toxicity to animals. Immunohistochemistry analysis confirmed in vitro findings such as downregulation of AR/pAKT signaling in UroA-treated C4-2B tumors, which suggests that UroA may be a potent chemo-preventive and therapeutic agent for CRPC.

Chronic exposure to cadmium is known to be a risk factor for human prostate cancer. Despite over-whelming evidence of cadmium causing carcinogenicity in humans, the specific underlying molecular mechanisms that govern metal-induced cellular transformation remain unclear. Acute exposure (up to 72 hr) to cadmium induces apoptosis in normal prostate epithelial cells (RWPE-1), while chronic exposure (>1 year) transforms these cells to a malignant phenotype (cadmium-transformed prostate epithelial cells; CTPE). Increased expression of autophagy-regulated genes; Plac8, LC3B and Lamp-1; in CTPE cells was associated with cadmium-induced transformation. Increased expression of Plac8, a regulator of autophagosome/autolysosome fusion, facilitates the pro-survival function of autophagy and upregulation of pAKT((ser473)) and NF-κβ, to allow CTPE to proliferate. Likewise, inhibition of Plac8 suppresses CTPE cell growth. Additionally, overexpression of Plac8 in RWPE-1 cells induces resistance to cadmium toxicity. Pharmacological inhibitors and an inducer of autophagy failed to affect Plac8 expression and CTPE cell viability, suggesting a unique role for Plac8 in cadmium-induced prostate epithelial cell transformation. These results support a role for Plac8 as an essential component in the cadmium-induced transformation of normal prostate epithelial cells to a cancerous state.

Background: Cadmium, an established carcinogen, is a risk factor for prostate cancer. Induction of autophagy is a prerequisite for cadmium-induced transformation and metastasis. The ability of Psoralidin (Pso), a non-toxic, orally bioavailable compound to inhibit cadmium-induced autophagy to prevent prostate cancer was investigated. Methods: Psoralidin was studied using cadmium-transformed prostate epithelial cells (CTPE), which exhibit high proliferative, invasive and colony forming abilities. Gene and protein expression were evaluated by qPCR, western blot, immunohistochemistry and immunofluorescence. Xenograft models were used to study the chemopreventive effects in vivo. Results: Cadmium-transformed prostate epithelial cells were treated with Pso resulting in growth inhibition, without causing toxicity to normal prostate epithelial cells (RWPE-1). Psoralidin-treatment of CTPE cells inhibited the expression of Placenta Specific 8, a lysosomal protein essential for autophagosome and autolysosome fusion, which resulted in growth inhibition. Additionally, Pso treatment caused decreased expression of pro-survival signalling proteins, NFκB and Bcl2, and increased expression of apoptotic genes. In vivo, Pso effectively suppressed CTPE xenografts growth, without any observable toxicity. Tumours from Pso-treated animals showed decreased autophagic morphology, mesenchymal markers expression and increased epithelial protein expression. Conclusions: These results confirm that inhibition of autophagy by Pso plays an important role in the chemoprevention of cadmium-induced prostate carcinogenesis.

We have previously reported that high aldehyde dehydrogenase (ALDH) enzyme activity in breast cancer cells results in breast cancer stem cell (BCSC) properties by upregualting Notch-1 and epithelial mesenchymal markers. This results in chemoresistance in breast cancer. Here, we examined the functional and clinical significance of ALDH expression by measuring the ALDH levels in breast cancer tissues by immunohistochemistry. There was a significantly higher ALDH expression in higher grade breast cancer tumor tissues (Grade- II and III) versus normal breast tissues. Injection of BCSC (ALDH+ and CD44+/CD22-) cells resulted in aggressive tumor growth in athymic mice versus ALDH- cells. The ALDH+ and CD44+/CD22- tumors grow rapidly and are larger than ALDH- tumors which were slow growing and smaller. Molecularly, ALDH+ tumors expressed higher expression of Notch-1 and EMT markers than ALDH- tumors. Oral administration of the naturally occurring Psoralidin (Pso, 25 mg/kg of body weight) significantly inhibited the growth in ALDH+ and ALDH- tumors as well. Psoralidin inhibited Notch-1 mediated EMT activation in ALDH+ and ALDH- tumors-this confirms our in vitro findings. Our results suggest that Notch-1 could be an attractive target and inhibition of Notch-1 by Psoralidin may prevent pathogenesis of breast cancer as well as metastasis. This article is protected by copyright. All rights reserved

Earlier, we reported that chronic cadmium (Cd)-exposure to prostate epithelial (RWPE-1) cells causes defective autophagy, which leads to the transformation of a malignant phenotype in both in vitro and in vivo models. However, the upstream events responsible for defective autophagy are yet to be delineated. The present study suggests that chronic Cd exposure induces endoplasmic reticulum (ER) stress that triggers the phosphorylation of stress transducers [protein kinase R-like ER Kinase- (PERK), eukaryotic translation initiation factor 2-alpha- (eIF2-α) and Activating Transcription Factor 4 -(ATF-4)], resulting in defective autophagy that protects Cd-exposed RWPE-1 cells. On the other hand, inhibition of the ATF4 stress inducer by siRNA blocked the Cd-induced defective autophagy in transforming cells. While dissecting the upstream activators of ER stress, we found that increased expression of reactive oxygen species (ROS) is responsible for ER stress in Cd-exposed RWPE-1 cells. Overexpression of antioxidants (SOD1/SOD2) mitigates Cd-induced ROS that results in inhibition of ER stress and autophagy in prostate epithelial cells. These results suggest that the induction of ROS and subsequent ER stress are responsible for defective autophagy in Cd-induced transformation in prostate epithelial cells.

INTRODUCTION AND OBJECTIVES:The role of androgen receptor (AR) signaling in bladder cancer (BCa) is not fully characterized. This study aimed to delineate the role of AR signaling in BCa and to det...

Purpose The role of androgen receptor (AR) signaling in bladder cancer (BCa) is not fully characterized. This study aimed to delineate the role of AR signaling in BCa and to determine whether the combination of AR inhibitor, Enzalutamide (Enz), and Cisplatin (Cis) efficiently inhibit the growth of BCa cells. Methods AR expression was determined in 89 human urothelial BCa specimens by immunohistochemistry. A panel of BCa cell lines was treated with Cis, Enz, or a combination of both (Enz + Cis). We determined the expression of AR, changes in apoptotic signaling, DNA damage, and analyzed effect on epithelial mesenchymal transformation markers. Result AR expression was detected in 61.4% of tumors from male BCa patients. Inhibition of AR signaling by Enz effectively inhibited the growth of AR+ BCa cells by inducing apoptosis (26%) in AR+ TCCSUP (P = 0.0201) and J82 (15%, P = 0.0386) cells. Interestingly, Enz + Cis synergistically inhibited the proliferation of BCa cells even at low concentrations by inducing proapoptotic signaling in AR+ BCa cells. Invasive and migratory potential of TCCSUP and J82 cells were reduced with Enz + Cis treatment, and associated with down-regulation of mesenchymal markers. Conclusions A high percentage of the bladder tumors from male patients in our cohort expressed AR. The combination of Enz and Cis synergistically inhibited growth of BCa cells more efficiently than single agent alone. This supports the rationale for future investigation of AR antagonists in combination with standard chemotherapy in MIBC.

Advancements in science and technology lead to the development of different sensors that measure precipitation, an intrinsic component of the hydrological cycle. As conventional rainfall measurement is tedious, satellite precipitation products are being developed. In the current study, the newly released ECMWF (European Centre for Medium-Range Weather Forecasts) ReAnalysis (ERA-5) along with the Climate Hazards Group InfraRed Precipitation with Station (CHIRPS) dataset was statistically evaluated against Indian Meteorological Department (IMD) gridded data and was employed for streamflow simulations using the Soil and Water Assessment Tool (SWAT) model in five contrasting climatic regions of India. Distinct calibration scenarios were developed to test the performance of these reanalysis datasets for simulating streamflow. From categorical and continuous statistical results, ERA-5 exhibited better performance in detecting low (0–5 mm/h) and medium intensity (6–25 mm/h) rainfall, whereas CHIRPS manifested better performance in detecting high intensity rainfall events (>25 mm/h). CHIRPS proved to be effective in simulating streamflows in three out of five basins, whereas IMD exhibited better performance in the other two basins leaving ERA-5 with poor performance for streamflow simulations in all the basins. It was also observed that Satellite Precipitation Products (SPP's) are accurate in humid and tropical regions when compared to arid and semi-arid regions. The worst performance was exhibited in the Ponnaiyar river basin by SPP's and hence bias correction and integration techniques were applied to improve the performance of streamflow simulations further. The novel integration of SPP employing nudging scheme yielded better streamflow simulations when forced into SWAT hydrological model compared to the streamflow simulations obtained when loaded with bias-corrected and raw datasets. The implemented nudging scheme improved the performance of streamflow simulations and hence can be implemented in any basin that is ungauged or resulting in poor hydrological modeling performance when employed with one SPP. The adopted nudging scheme will be highly useful to generate long-term consistent precipitation records in an ungauged or poorly gauged river basin.

Mouse Double Minute homolog 4 (MDM4) gene up-regulation often occurs in human hepatocellular carcinoma (HCC), but the molecular mechanisms responsible for its induction remain poorly understood. Here we investigated the role of the phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/AKT/mTOR) axis in the regulation of MDM4 levels in HCC. The activity of MDM4 and the PI3K/AKT/mTOR pathway was modulated in human HCC cell lines by way of silencing and overexpression experiments. Expression of main pathway components was analyzed in an AKT mouse model and human HCCs. MDM4 inhibition resulted in growth restraint of HCC cell lines both in vitro and in vivo. Inhibition of the PI3K-AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells and reactivated p53-dependent transcription. Deubiquitination by ubiquitin-specific protease 2a and AKT-mediated phosphorylation protected MDM4 from proteasomal degradation and increased its protein stability. The eukaryotic elongation factor 1A2 (EEF1A2) was identified as an upstream inducer of PI3K supporting MDM4 stabilization. Also, we detected MDM4 protein up-regulation in an AKT mouse model and a strong correlation between the expression of EEF1A2, activated/phosphorylated AKT, and MDM4 in human HCC (each rho > 0.8, P

Aims The Far Upstream Element [FUSE] Binding Protein 1 (FUBP1) regulates target genes, such as the cell cycle regulators MYC and p21. FUBP1 is up-regulated in many tumours and acts as an oncoprotein by stimulating proliferation and inhibiting apoptosis. Recently, FUBP1 mutations were identified in approximately 15% of oligodendrogliomas. To date, all reported FUBP1 mutations have been predicted to inactivate FUBP1, which suggests that in contrast to most other tumours FUBP1 may act as a tumour suppressor in oligodendrogliomas. Methods As no data are currently available concerning FUBP1 protein levels in gliomas, we examined the FUBP1 expression profiles of human glial tumours by immunohistochemistry and immunofluorescence. We analysed FUBP1 expression related to morphological differentiation, IDH1 and FUBP1 mutation status, 1p/19q loss of heterozygosity (LOH) as well as proliferation rate. Results Our findings demonstrate that FUBP1 expression levels are increased in all glioma subtypes as compared with normal central nervous system (CNS) control tissue and are associated with increased proliferation. In contrast, FUBP1 immunonegativity predicted FUBP1 mutation with a sensitivity of 100% and a specificity of 90% in our cohort and was associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity (LOH). Using this approach, we detected a to-date undescribed FUBP1 mutation in an oligodendroglioma. Conclusion In summary, our data indicate an association between of FUBP1 expression and proliferation in gliomas. Furthermore, our findings present FUBP1 immunohistochemical analysis as a helpful additional tool for neuropathological glioma diagnostics predicting FUBP1 mutation.

Introduction: Cadmium (Cd) exposure is associated with an increased risk of prostate cancer (CaP). Previously we reported that Cd induces Plac8 expression may be responsible for defective autophagy which facilitates the transformation of prostate epithelial cells. The goal of this study is to decipher the molecular signaling responsible for defective autophagy by plac8 and whether inhibition of plac8 facilitates pro-apoptotic machinery in chronically exposed -Cd in prostate epithelial cells. Methods: Overexpression or silencing Plac8 in chronically exposed Cd in RWPE-1 cells or cadmium, transformed prostate epithelial (CTPE) cells were subjected to cell viability, apoptosis, autophagy functional studies, Western blot and in vivo analyses. For statistical analysis, data were analyzed using Student’s‘t’ test with a p-value less than 0.05 considered significant. Results: Our results suggest that during cellular transformation, Cd exposure induced Plac8 expression, which caused defective-autophagy that resulted in the proliferation of damaged cells. Inhibition of Plac8 resulted in suppression of pro-survival machinery and concomitantly induced severe endoplasmic reticulum (ER) stress that resulted in caspase-mediated cell death in Cd-exposed cells. More specifically, inhibition of Plac8 by shRNA resulted in induction of ER stress markers (PREK/ATF4) which facilitate caspase-mediated cell death in Cd- transformed prostate epithelial cells. Activation of NFKB is one of the downstream events of Plac8 that transcriptionally regulates NFKB activation in Cd-transformed cells. We identified, Plac8 binding sites in NFKB promoter. Hence inhibition of plac8 resulted in the complete shutdown of NFKB mediated pro-survival signaling in Cd-transformed prostate epithelial cells. While analyzing autophagy signaling network, we found induction of Atg family proteins, however, in the absence of Plac8, the induction of autophagy fails to protect the Cd-transformed cells. Similar results were observed in plac8 silenced cd-transformed cells in xenotransplanted mice, where Cd-transformed cells induced aggressive tumors, and Plac8 knockdown significantly inhibited the tumor growth. While ATF4, PLac8, NFKB were not expressed in normal human prostate tissues, their expression was significantly increased in BPH followed by CaP specimens; normal prostate < BPH << Gleason 6-7 CaP < Gleason 8-10 CaP. Cd levels were much higher in tumor tissues (21.43 μg/g) of CaP patients than in healthy controls (1.12 μg/g). The levels also correlated with Plac8 expression. These results demonstrate the clinical significance of Cd levels and of Plac8 expression as plausible drivers of Cd-induced transformation in prostate carcinogenesis. Conclusions: These results suggest that Plac8 may be an essential component in the Cd-induced transformation of normal prostate epithelial cells to a cancerous state. Citation Format: Venkatesh Kolluru, Ashish Tyagi, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Inhibition of autophagy (Plac8) prevents malignant transformation of cadmium induced prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4277.

Introduction: Inactivating p53 mutations contribute to tumor progression and treatment-resistance, resulting in poor patient survival in colorectal cancer patients (CRC). The goal of this study is to identify novel small molecules that therapeutically target mutant p53 in CRC. Methods: We analyzed the effects of small molecule (ASR458) on p53-wild type (p53-wt; HCT116) and mutant p53 (p53-mut; SW620) colon cancer cells by phenotypic, molecular and in vivo assays. Results: ASR458 treatment significantly inhibited the proliferation of both HCT116 and SW620 cells at nM concentrations. In p53-wt HCT116 cells, ASR458 caused induction of p53 that resulted in caspase-mediated cell death in both in vitro and in vivo models. On the contrary, ASR458 treatment induced ER-stress signaling (i.e., phosphorylation of ERK and eIF2-α) in p53-mut SW620 cells, which triggered ATF4 activation and subsequent induction of cascade of autophagy events (Atg family proteins, LC3B and Lamp1), causing autophagy-mediated cell death. Silencing ER stress marker ATF-4, a key regulator of autophagy, caused resistance to ASR458 and abrogated autophagy signaling in SW620 cells. This suggested that induction of ER-stress is critical for the cytotoxic effects of ASR458 in p53-mut CRC. Preliminary knockdown studies indicate that silencing of ER markers causes resistance to ASR458 in vitro, further confirming ER-stress as the mechanism of ASR458 action in p53-mut CRC. ASR458 significantly inhibited the growth of SW620 tumors in xenograft. Tissue analysis confirmed the ER-stress signaling observed in vitro. Conclusion: Our results demonstrate ASR458 as a potential therapeutic agent with distinct targets in p53-wt and p53-mut CRC. This study also suggests that ATF4 mediated autophagy in unmanaged ER stress can reduce CRC pathogenesis. Further investigation into the pharmacokinetics and pharmacodynamics of ASR458 should help clinical translation of this agent. Citation Format: Ashish Tyagi, Balaji chandrasekaran, Venkatesh Kolluru, Becca V. Baby, Alatassi Houda, Srinivasa Ramisetti, Arun Sharma, Murali Ankem, Chendil Damodaran. Endoplasmic reticulum (ER), a potential therapeutic target for mutant p53 colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4266.

The transcription factor, androgen receptor (AR) is a key driver and plays an important role in prostate cancer. Androgen ablation therapy and brachytherapy remains the standard treatment of advanced prostate cancer, but unfortunately, it is not curative, and eventually the disease returns as lethal castration-resistant prostate cancer (CRPC). Previously we have reported that Urolithin A (Uro A), a natural compound, inhibits AR signaling and suppresses the growth of CRPC. Now, we have developed a series of pro-drug conjugates of Uro A and our initial structure-activity relationship (SAR) studies led to the identification of three small molecules as lead compounds. Hence, we investigated the effects of all three compounds and dissected their molecular mechanism in cell culture and mouse models of CRPC. All the three compounds more effectively inhibited the growth CRPC cell lines than the parent compound Uro A. Based on their IC50 concentration; we identified a novel compound (ASR-600) that demonstrated better efficacy by inhibiting AR signaling in CRPC cell lines than the parental compound Uro A. The model system and molecular dynamics (MD) stimulation studies suggest that ASR600 bind to the ligand-binding domains of AR and blocks the conformation changes, that allows AR to phosphorylate and degrade in the cytosol. Further, our thermal shift assay confirmed ASR 600 binds to AR in presence and absence of Dihydrotestostreone. Our ongoing in vivo studies, may suggest whether oral administration of ASR-600 effectively inhibits the tumor growth raised from CRPC cell lines (C4-2B, 22RV1 and enzalutamide resistance C4-2B) in xenografted mice. These observations provide a rationale for devising novel therapeutic agent based on ASR 600 for the treatment of CRPC Citation Format: Balaji Chandrasekaran, Ashish Tyagi, Venkatesh Kolluru, Aakash M. Vadhanam, Becca von Baby, Suresh Gorle, Suman Sirimulla, Srinivasa R. Ramisetti, Arun K. Sharma, Murali K Ankem, Chendil damodaran. Developing small molecule inhibitors that target androgen receptor signaling in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2598.

Introduction: Earlier we reported that aberrant overexpression of Notch1 in colon cancer patients is responsible for aggressiveness and progression of the disease. Current, therapeutic interventions of notch1 inhibitors that binds catalytic unit of γ-secretase and disrupts the cleavage activity cause gastrointestinal toxicity in clinical trial. Therefore, our study aims to develop a small molecule inhibitor, which inhibits Notch1 signaling but binds outside of γ-secretase’s catalytic unit thereby substantially reducing associated toxicity attributes. Through structure-activity relationship (SAR) studies based on naturally occurring Withaferin A (WA) structure, we have recently identified a WA-difuroate analog ASR458 as one such small molecule. Methods: To analyze the effect of ASR490 on HCT116 and HCT116/Notch1 cells, we performed phenotypic, western blot and in vivo analysis. Results: Notch1 overexpressing CRC cells (HCT/Notch1) showed aggressive cell growth (42%) as compared to HCT116 cells, however, ASR490 treatment inhibited cell growth in both HCT116 (IC50: 600 nM) as well as HCT/Notch1 cells (IC50: 850 nM). ASR458 significantly inhibited Notch1 expression and its downstream events in both the transfected cell lines. As a result, we observed shutdown of pro-survival machinery and induction of pro-apoptotic events (Bax, caspases and PARP) resulting in apoptotic cell death in ASR456 treated HCT116 and Notch1/HCT116 cells. Since Notch1 is responsible for epithelial mesenchyme transition (EMT); we analyzed EMT drivers and readout invasion/migration assays. ASR458 treatment reversed the mesenchymal characteristics by restoring the function of E-cadherin, which resulted in the suppression of invasion and migration in readout assays of HCT116 transfectants. Our ongoing Xenotransplanted mice experiments may suggest whether ASR458 inhibits the growth of HCT116 and HCT116/Notch1 tumors. Conclusion: Restrained gastrointestinal toxicity of Notch1 targeting therapeutics is achievable with novel small molecules displaying a differential binding affinity to γ-secretase. ASR458 inhibits cell growth in Notch1 overexpressing cells and abrogates Notch1 induced EMT. Ongoing molecular docking and in vivo studies may confirm ASR458 binding sites in γ-secretase and validate our in vitro findings respectively. Citation Format: Chendil Damodaran, Ashish Tyagi, Balaji Chandrasekaran, Venkatesh Kolluru, Ibrahim Jojua, Srinivasa Ramisetti, Arun K. Sharma, Murali K. Ankem. A novel small molecule inhibitor to suppress Notch1 activation in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2014.

Background Prostate-specific antigen screening for prostate cancer (CaP) remains controversial. This study establishes the role of microRNA 301a (miR-301a) as a supplemental biomarker that can distinguish between patients with benign prostate hyperplasia and clinically significant CaP. We evaluate the ability of miR-301a to predict the adverse pathology of CaP. Methods In the first cohort, serum and prostate tumor samples were obtained from thirteen patients with Benign prostate hyperplasia (BPH), twelve patients with Gleason 6, and sixteen patients with Gleason 7 prostate adenocarcinoma. In the second cohort, 40 prostatectomy cases were selected (BPH:12, Gleason 6:12 and Gleason 7:16). MiRNA was extracted from serum and tumor samples. Quantitative reverse transcription-polymerase chain reaction was performed for detection of miR-301a. To understand the molecular role of miR-301a, we performed cell viability, Western blots, promoter analysis, overexpression, and silencing studies in BPH and DU-145 cell lines. Results MiR-301a demonstrated a significantly higher expression in both serum and tumor tissue in patients with CaP when compared to patients with BPH (P = 0.011 and 0.013 for serum and tissue expression, respectively). Expression of miR-301a in prostatectomy specimens correlated with increased Gleason score. We demonstrated that miR-301a inhibited the pro-apoptotic function of RUNX3, and activated ROCK1-mediated pro-survival signal in CaP. Silencing miR-301a initiated the pro-apoptotic function of RUNX3 by inhibiting ROCK1 expression in CaP cells. Conclusions Expression of miR-301a could be a valuable adjunct tool for stratifying patients with elevated prostate-specific antigen, as well as those diagnosed with CaP. Including the miR-301a as an additional variable in MSKCC post-prostatectomy nomogram improved its ability in facilitating clinical decision-making.

// Suman Suman 1 , Trinath P. Das 1 , Jim Moselhy 1 , Deeksha Pal 1 , Venkatesh Kolluru 1 , Houda Alatassi 2 , Murali K. Ankem 1 Chendil Damodaran 1 1 Department of Urology, University of Louisville, KY, USA 2 Department of Pathology, University of Louisville, KY, USA Correspondence to: Chendil Damodaran, email: chendil.damodaran@louisville.edu Keywords: dietary agents, chemoprevention, prostate cancer Received: April 28, 2016 Accepted: June 13, 2016 Published: July 20, 2016 ABSTRACT We previously reported that withaferin A (WA), a natural compound, deters prostate cancer by inhibiting AKT while inducing apoptosis. In the current study, we examined its chemopreventive efficacy against carcinogenesis in the prostate using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Two distinct sets of experiments were conducted. To determine whether WA delays tumor progression, it was given before cancer onset, at week 6, and until week 44. To determine its effect after the onset of prostate cancer, it was given from weeks 12 to 35. In both strategies, oral administration of WA effectively suppressed tumor burden when compared to vehicle-treated animals. No toxicity was seen in treated animals at gross pathological examination. Western blot analysis and immunohistochemistry of tumor sections revealed that in TRAMP controls, AKT and pAKT were highly expressed while nuclear FOXO3a and Par-4 were downregulated. On the contrary, treated mice showed inhibition of AKT signaling and activation of FOX03a-Par-4-induced cell death. They also displayed inhibition of mesenchymal markers such as β-catenin, vimentin, and snail as well as upregulation of E-cadherin. Because expressions of the angiogenic markers factor VIII and retic were downregulated, an anti-angiogenic role of WA is suggested. Overall, our results suggest that WA could be a promising anti-cancer agent that effectively inhibits carcinogenesis of the prostate.

We have previously reported that high aldehyde dehydrogenase (ALDH) enzyme activity in breast cancer cells results in breast cancer stem cell (BCSC) properties by upregualting Notch-1 and epithelial mesenchymal markers. This results in chemoresistance in breast cancer. Here, we examined the functional and clinical significance of ALDH expression by measuring the ALDH levels in breast cancer tissues by immunohistochemistry. There was a significantly higher ALDH expression in higher grade breast cancer tumor tissues (Grade- II and III) versus normal breast tissues. Injection of BCSC (ALDH(+) and CD44(+)/CD22(−)) cells resulted in aggressive tumor growth in athymic mice versus ALDH(−) cells. The ALDH(+) and CD44(+)/CD22(−) tumors grow rapidly and are larger than ALDH(−) tumors which were slow growing and smaller. Molecularly, ALDH(+) tumors expressed higher expression of Notch-1 and EMT markers than ALDH(−) tumors. Oral administration of the naturally occurring Psoralidin (Pso, 25 mg/kg of body weight) significantly inhibited the growth in ALDH(+) and ALDH(−) tumors as well. Psoralidin inhibited Notch-1 mediated EMT activation in ALDH(+) and ALDH(−) tumors-this confirms our in vitro findings. Our results suggest that Notch-1 could be an attractive target and inhibition of Notch-1 by Psoralidin may prevent pathogenesis of breast cancer as well as metastasis.

Purpose The diagnosis and treatment of prostate cancer (CaP) continues to be challenging, as prostate-specific antigen (PSA) appears to be overly sensitive and biopsy is the only reliable method for confirmation. Hence, the goal of the study is to identify a biomarker that could distinguish malignant cancer from benign prostatic hyperplasia (BPH) during the early diagnosis of the disease. Materials and methods A total of 75 formalin fixed paraffin embedded (FFPE) with matching controls, 4 paired metastatic tumors, 6 fresh tumor tissues and BPH (13 cases) with their clinical diagnosis were selected for this study. Prostate cancer cell lines and normal prostate epithelial cell lines were obtained from ATCC and subjected to phenotypic analysis. Results We observed significant differential expression of miR-301a in CaP samples in comparison to BPH and adjacent benign samples. The overexpression of miR-301a activates the invasion/migration of CaP cells. In contrast, silencing miR-301a expression inhibited the colony-forming ability, adhesion, invasion and migration of CaP cells. Similarly, the overexpression of miR-301a increased cell motility in normal RWPE-1 prostate epithelial cells. Our results suggest that miR-301a is differentially expressed between BPH and CaP specimens and that the expression of miR-301a correlates with biochemical recurrence and/or metastasis in CaP patients. Conclusions The expression of miR-301a could be a potential marker for metastasis in CaP patients. Detecting miR-301a expression during diagnosis will avoid wait and watch timelines, thus preventing morbidity.

Introduction: Exposure to cadmium (Cd) is associated with a spectrum of human pathogenesis including the prostate cancer (CaP). A clear dose-response relation between Cd-exposure and CaP have been reported in men exposed to Cd. However, the molecular mechanism underlying the malignant cell transformation following Cd exposure is yet to be determined. One of the possible mechanisms is that Cd causes endoplasmic reticulum (ER) stress, which further induces defective autophagy that plays a cytoprotective role in response to the misfolded and unfolded proteins that are formed during cellular transformation. Hence, the goal of this study is to investigate the underlying mechanism of how Cd causes malignant cell transformation (from normal to cancer cells) and on the development of tumorigenesis by the Cd-transformed cells. Methods: Normal prostate epithelial cells (RWPE-1) and Cd (10µM)-transforming prostate epithelial cells and cadmium-transformed prostate epithelial cells (CTPE) were utilized. Overexpression and/or silence ER-sensors, EGFR, and p62 were performed in above mention cell lines and subjected to cell viability, apoptosis, autophagy functional studies and Western blot analyses. For statistical analysis, data were analyzed using Student's ‘t' test with a p-value less than 0.05 considered significant. Results: Our preliminary results suggest that during cellular transformation, Cd exposure induced ER-stress, which triggered the phosphorylation of stress transducers including protein kinase R-like ER Kinase (PERK) and e1F2-α (eukaryotic translation initiation factor 2A-alpha). Phosphorylation resulted in the activation of ATF4 (Activating Transcription Factor 4) and autophagy induction thus enhancing protection of Cd-damaged cells. Further, inhibition of stress inducers (ATF4) or p62 by siRNA blocked the Cd-induced defective autophagy resulted in growth inhibition in transforming cells. Interestingly, in Cd-transformed cells, blocking EGFR activation by siRNA or pharmacological inhibitors significantly inhibited the growth, but not in the transforming cells suggesting that EGFR activation plays a critical role only after cellular transformation. Further, xenograft tumor tissues generated by Cd-transformed cells expressed high levels of ATF-4, EGFR, p62 and LC3B in correlation with in vitro findings. Moreover, increased expression of the proteins (ATF-4, EGFR, p62, and LC3B) in human CaP specimens correlates with Gleason sum in comparison with benign prostatic hyperplasia and “normal” adjacent tissues. Conclusions: The results suggest that ER stress responsible for the defective autophagy in Cd-induced transformation. This study highlights the better understanding of the complex interrelationship among prostate cancer phenotypes and the molecular, cellular, biochemical, and pathological changes associated with Cd and prostate cancer. Citation Format: Venkatesh Kolluru, Ashish Tyagi, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Cadmium-induced endoplasmic reticulum stress causes defective autophagy in human prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1328.

Treatment of prostate cancer is still clinically challenging due to lack of reliable markers for diagnosis and prognosis. Serum prostate specific antigen (PSA) is of limited clinical utility due to poor sensitivity and specificity. A large number of false positives (50%) with traditional PSA testing lead many patients to undergoing unnecessary prostate biopsy and exposure to unnecessary risk of complications. So the goal of this study is to identify a reliable, non-invasive biomarker that can distinguish patients with benign, indolent and aggressive prostate cancer in various clinical settings. MicroRNAs (miRNAs) are small noncoding (18 to 28 nucleotides) RNA molecules that are present in all human cells, and their expression patterns are correlated with many cancer types, including CaP. In our results, we found a significant differential expression (p=0.013) of miR-301a in both tumor tissues (Gleason-6 and -7) and serum samples in comparison to benign prostatic hyperplasia (BPH) or adjacent benign samples. We observed a negative correlation between miR-301a and RUNX3 expression in human CaP samples suggesting tumor suppressive role RUNX3 might be compromised in CaP. To determine MiR-301a regulation on RUNX3, we evaluated miR-301a and RUNX3 protein in panel of prostate cancer cell lines, normal prostate epithelial cells and Benign Prostate Hyperplasia (BPH). Similar results such as inverse correlation between miR-301a and RUNX were found in cell culture models. Also, over expression of miR-301a down regulates RUNX3 activation in prostate cancer cell lines and silencing miR-301a expression reverts RUNX3 activation in BPH cells. While evaluating the reporter activity of RUNX3 either by co-transfecting with mimic or inhibitor of miR-301a in BPH and prostate cancer cells: over expression of miR-301a down regulated RUNX3 reporter activity which corresponded with RUNX3 protein expression. Similarly, silencing miR-301a reverted RUNX3 promoter activity and protein expression suggesting RUNX3 is a direct target of miR-301a in CaP cells. Silencing miR-301a reverted the pro-apoptotic function of RUNX3, results in reduced colony formation, adhesion, invasion and migration of CaP cells. Investigating the mechanistic link miR-301a and RUNX3 may explore whether it could facilitate clinical decision making such as the decision to proceed with early surgery rather than active surveillance for intermediate risk prostate cancer patients deemed to be at high risk of progression. Citation Format: Venkatesh Kolluru, Balaji Chandrasekhar, Collin McKenzie, Houda Alatassi, Murali Ankem, Chendil Damodaran. Inhibition of tumor suppressor function of RUNX3 by miR-301a facilitates the progression of castration resistant prostate cancer [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 5445. doi:10.1158/1538-7445.AM2017-5445

Lung cancer remains a leading public health problem which is evidenced by its increasing death rate. Platinum-based chemotherapy is the first-line of treatment for patients in advanced stages of non-small cell lung cancer (NSCLC), however the success rates are not quite impressive. Hence, developing individualized treatment strategies for metastatic lung cancer gains momentum, such as Tyrosine kinase inhibitors erlotinib and gefitinib, or ALK inhibitors ceritinib and alectinib are commonly used in the clinic. Recent reports suggest that pAKT(ser473) is highly expressed in NSCLC and higher nuclear expression of pAKT correlated with poor prognosis and an independent prognostic marker for survival. Our lab is interested to develop novel small molecules which specifically inhibit AKT signaling in NSCLC. Structure-activity relationship (SAR) studies in our laboratory have recently ideintifed one such compound, AKS-407, that effectively inhibited cell growth at nanomolar concentration in NSCLC cell lines (A549 and H460; 250nM). Molecular studies revealed AKS-407 inhibited AKT signaling by down regulating pAKT(ser473) expression and downstream events including NFκB activation, BCl-2 expression in both the cell lines. As signaling through AKT regulates epithelial-mesenchymal transition (EMT) in NSCLC, we determined the effect of AKS-407 on EMT phenotype on NSCLC cells. Treatment of AKS-407 inhibited the mesenchymal markers includes snail, MMP9, N-cadherin, β-catenin and vimentin expression that resulted in blocking invasion and migration of A549 and H-460 cells. These results suggest AKS-407 to be a promising small molecule targeting AKT signaling pathway which remains an important target for the development of effective treatment of metastatic NSCLC. Validating in-vivo efficacy of this potential drug candidate would further support our overall goal of the study. Citation Format: Balaji Chandrasekaran, Deeksha Pal, Venkatesh Kolluru, Srinivasa R. Ramisetti, Arun K. Sharma, Murali Ankem, Chendil Damodaran. Developing small molecule therapeutics to target AKT signaling in non-small cell lung cancer [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 371. doi:10.1158/1538-7445.AM2017-371

We recently showed that two different ALDH+ and CD44+/CD24-/low breast cancer stem cells (BSCSs) exhibited stem cell characteristics that include self-renewal, extensive proliferation, the ability to form non-adherent spherical clusters, chemotherapy resistance and high Notch1 expression. We have identified a compound compound: 6-(3-methylbut-2-enyl) coumestrol (Pso) and treatment with Pso resulted in growth inhibition and an EMT phenotype in both BCSCs and BC cells. Oral Pso administration at physiologically achievable doses (25 mg/kg/BW) suppressed the growth of BCSCs and BC xenografts without toxicity. In the current studies, we identified several novel Pso-derived analogs that may be more potent than the parent compound. One such compound, 1-methoxyphaseollidin (1MP), obtained via three main functional group changes: (i) translocation of the isoprenyl moiety from the phenyl ring fused to the pyran ring (as in Pso) to the phenyl ring adjacent to the furan ring, (ii) removal of the carbonyl group from the pyran ring, and (iii) introduction of a methoxy group at the 1-position, inhibited Notch1 activity and growth of both BSCS and BC cells at nM concentration (IC50: 300nM), which is 100 times more potently than Pso in cell culture models. Molecular studies suggest that 1MP inhibits Notch signaling pathways (Hes1, Hey1 and Presenilin) in both BCSC and BC cells. Further, downregulation of AKT signaling (pAKT (S473), p65 and BCl-2 were seen in 1MP treated cells. Docking studies suggest that 1MP binds outside of the catalytic unit of γ-secretase and induces a conformational change, resulting in Notch1 inhibition in both BCSCs and BC cells. More importantly, administration of 1MP significantly inhibited the growth of BCSC and BC tumors without causing gastrointestinal toxicity in tumor-bearing mice. H&E staining suggests that 1MP treated tumors show infiltrate to margins are less as compared to vehicle treated mice’s. We believe targeting notch1 signaling and optimizing 1MP could be an effective therapeutic strategy for treating breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Venkatesh Kolluru, Deeksha Pal, Becca Baby, Houda Alatassi, Arun Kumar Sharma, Murali Ankem, Chendil Damodaran. 1-Methoxyphaseollidin: Novel gamma secretase inhibitor targeting notch-1 signaling in breast cancer stem 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 2903. doi:10.1158/1538-7445.AM2017-2903

Dupuytren's disease (DD) is a fibroproliferative disorder characterized by aberrant proliferation of myofibroblasts, the source of which remains unknown. Recent studies indicate that circulating and tissue-resident mesenchymal stem cells (MSCs) can differentiate into myofibroblasts. Therefore, the aim of this study was to profile MSCs from phenotypically distinct DD sites including cord, nodule, skin overlying nodule (SON), and perinodular fat (PNF) compared with unaffected internal controls, that is, distant palmar fat (DPF) and transverse palmar fascia (Skoog's fibers) as well as external control carpal tunnel (CT) tissue including skin, fat, and fascia. Freshly isolated primary fibroblasts as well as cells grown up to passage 5 (P5) from DD (n=27) and CT (n=14) samples were analyzed for the presence of established MSC markers CD73, CD90, and CD105 and absence of hematopoietic marker CD34 using fluorescence-activated cell sorting, in-cell quantitative western blotting, immunohistochemistry, and immunocytochemistry. Freshly isolated cells from SON, PNF, and cord biopsies had a higher number of CD34(-)73(+)90(+)105(+) cells compared with Skoog's fibers and CT controls. P3 cells obtained from all DD biopsies compared with CT samples differentiated into osteocytes, adipocytes, and chondrocytes. P3 cord and nodule cells expressed intense α-smooth muscle actin staining compared with skin and fat cells. Stem cell markers including stem cell factor, MSC-homing marker CXCR4, and Wnt/β-catenin downregulator Dkk-1 were all upregulated in SON and PNF compared with CT skin and CT fat, respectively, as shown by real-time quantitative polymerase chain reaction. However, osteogenic marker OSF-1 had a significantly higher expression in the PNF (P=0.002) and cord (P=0.01) compared with the nodule. In conclusion, we have shown the presence of MSCs in specific DD tissue phenotypes compared with internal and external control tissue. These findings provide preliminary support for a potential alternative source of disease myofibroblasts originating from sites such as SON and PNF as opposed to palmar fascia alone.