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Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy-resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Bone metastatic prostate cancer (BM-PCa) remains one of the most difficult cancers to treat due to the complex interactions of cancer and stromal cells. We previously showed that bone marrow neutrophils elicit an anti-tumor immune response against BM-PCa. Further, we demonstrated that BM-PCa induces neutrophil oxidative burst, which has previously been identified to promote primary tumor growth of other cancers, and a goal of this study was to define the importance of neutrophil oxidative burst in BM-PCa. To do this, we first examined the impact of depletion of reactive oxygen species (ROS), via systemic deletion of the main source of ROS in phagocytes, NADPH oxidase (Nox)2, which we found to suppress prostate tumor growth in bone. Further, using pharmacologic ROS inhibitors and Nox2-null neutrophils, we found that ROS depletion specifically suppresses growth of androgen-insensitive prostate cancer cells. Upon closer examination using bulk RNA sequencing analysis, we identified that metastatic prostate cancer induces neutrophil transcriptomic changes that activates pathways associated with response to oxidative stress. In tandem, prostate cancer cells resist neutrophil anti-tumor response via extracellular (i.e., regulation of neutrophils) and intracellular alterations of glutathione synthesis, the most potent cellular antioxidant. These findings demonstrate that BM-PCa thrive under oxidative stress conditions and such that regulation of ROS and glutathione programming could be leveraged for targeting of BM-PCa progression.

Neuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2's role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy.

Urothelial bladder cancer ranks among the 10 most frequently diagnosed cancers worldwide. In our previous study, the transmembrane protein neuropilin-2 (NRP2) emerged as a predictive marker in patients with bladder cancer. NRP2 consists of several splice variants, the most abundant of these, NRP2a and NRP2b, are reported to have different biological functions in lung cancer progression. For other cancer types, there are no published data on the role of these transcript variants in cancer progression and the clinical outcome. Here, we correlate NRP2 and its two most abundant transcript variants, NRP2A and NRP2B, with the clinical outcome using available genomic data with subsequent validation in our own cohort of patients with muscle-invasive bladder cancer. In addition to NRP2, NRP1 and the NRP ligands PDGFC and PDGFD were studied. Only NRP2A emerged as an independent prognostic marker for shorter cancer-specific survival in muscle-invasive bladder cancer in our cohort of 102 patients who underwent radical cystectomy between 2008 and 2014 with a median follow-up time of 82 months. Additionally, we demonstrate that high messenger expression of NRP2, NRP1, PDGFC and PDGFD associates with a more aggressive disease (i.e., a high T stage, positive lymph node status and reduced survival).

The presence of positive surgical margins confers an increased risk of biochemical relapse and need for salvage therapy in men undergoing radical prostatectomy. Image-guided surgery using near-infrared (NIR) fluorescent contrast agents is a potential method to detect remaining cancerous tissue. The objective of this study was to evaluate three hyaluronic acid (HA) nanoparticle (NP) formulations loaded with NIR fluorophore for their ability to contrast-enhance prostate cancer. HA was modified by conjugation with the hydrophobic ligand, aminopropyl-1-pyrenebutanamide to drive nanoparticle self-assembly. Indocyanine green (ICG) was physicochemically entrapped in the HA-NP, termed NanoICG. Alternatively, Cy7.5 was directly conjugated to amphiphilic HA, termed NanoCy7.5. NanoCy7.5 was synthesized with two HA molecular weights to determine the HA size contribution to delivery to PC3 prostate tumor xenografts. Contrast-enhancement of the tumors and relative biodistribution were assessed by a series of fluorescence imaging, image-guided surgery with spectroscopy, and microscopic techniques. Intravenously administered NanoICG improved tumor signal-to-noise ratio (SNR) at 24 h over ICG by 2.9-fold. NanoCy7.5 with 10 kDa and 100 kDa HA improved tumor SNR by 6.6- and 3.1-fold over Cy7.5 alone, respectively. The PC3 xenograft was clearly identified with the image-guided system providing increased contrast enhancement compared to surrounding tissue for NanoICG and NanoCy7.5 with 10 kDa HA. NIR fluorescence microscopy showed that Cy7.5 in NPs with 10 kDa HA were distributed throughout the tumor, while NanoCy7.5 with 100 kDa HA or NanoICG delivered dye mainly to the edge of the tumor. CD31 staining suggested that PC3 tumors are poorly vascularized. These studies demonstrate the efficacy of a panel of HA-derived NPs in identifying prostate tumors in vivo, and suggest that by tuning the structural properties of these NPs, optimized delivery can be achieved to poorly vascularized tumors. Statement of Significance We have demonstrated the potential of a panel of near-infrared fluorescent (NIRF) nanoparticles (NPs) for image-guided surgery in a prostate cancer xenograft model. Image-guided surgery and imaging of organs ex vivo showed greater tumor signal and contrast when mice were administered NIRF dyes that were covalently conjugated to (NanoCy7.510k-PBA) or physicochemically entrapped in (NanoICGPBA) hyaluronic acid (HA) NPs, compared to free dyes. These results show the potential to use these NPs as tools to detect the margins of tumors and to differentiate healthy and tumor tissue intraoperatively. Moreover, this project provides insight into selecting optimal formulation strategies for poorly vascularized tumors.

Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

Genetic abnormalities in components important for the folate cycle confer risk for various disorders since adequate folate turnover is necessary for normal methylation, gene expression and chromosome structure. However, the system has rarely been studied in children diagnosed with attention deficit hyperactivity disorder (ADHD). We hypothesized that ADHD related cognitive deficit could be attributed to abnormalities in the folate cycle and explored functional single nucleotide polymorphisms in methylenetetrahydrofolate dehydrogenase (rs2236225), reduced folate carrier (rs1051266), and methylenetetrahydrofolate reductase (rs1801131 and rs1801133) in families with ADHD probands (N = 185) and ethnically matched controls (N = 216) recruited following the DSM-IV. After obtaining informed written consent for participation, peripheral blood was collected for genomic DNA isolation and PCR-based analysis of target sites. Data obtained was analyzed by UNPHASED. Interaction between sites was analyzed by the multi dimensionality reduction (MDR) program. Genotypic frequencies of the Indian population were strikingly different from other ethnic groups. rs1801133 "T" allele showed biased transmission in female probands (p < 0.05). Significant difference in genotypic frequencies for female probands was also noticed. rs1801131 and rs1801133 showed an association with low intelligence quotient (IQ). MDR analysis exhibited independent effects and contribution of these sites to IQ, thus indicating a role of these genes in ADHD related cognitive deficit.

Intellectual disability (ID) is of major concern throughout the world, though in ~40% of cases etiology remains unknown (idiopathic ID or IID). Cognitive impairment and behavioral problems are of common occurrence in these subjects and dopamine is known to play an important role in regulating these traits. In the present study the role of functional polymorphisms in three dopaminergic genes, dopamine receptor D4 (DRD4: exon3 VNTR and rs1800955), dopamine transporter (DAT1: 3'UTR VNTR and intron8 VNTR) and catechol-O-methyl transferase (COMT: rs4680 and rs165599), was explored in IID. Probands (n=225), parents (n=298) and ethnically matched controls (n=175) were recruited following DSM-IV. Genotype data obtained was used for population- and family-based statistical analyses. Population-based analysis showed significant association of DRD4 exon3 VNTR 6R allele (P=0.01), DAT1 3'UTR VNTR lower repeat (6R and 7R) alleles (P0.02) and intron8 VNTR 5R allele (P=0.0012) with IID. Stratified analysis revealed significant association of these alleles (P0.05) with IID individuals exhibiting severe behavioral problems. On the other hand, preferential transmission of COMT rs4680 A allele and A-A haplotype (P0.05) was observed specifically in male IID probands without any behavioral problem. Markers failed to show any significant epistatic interaction by MDR analysis. Alleles showing positive association were all reported to confer suboptimal activity to the transcribed proteins. Therefore, an alteration in dopaminergic neurotransmission could be predicted that may lead to impairments in cognition and behavioral problems.

Down Syndrome (DS) patients suffer from cognitive dysfunction, depression, hyperactivity, irritability etc. Dopamine (DA) and serotonin (5HT) are known to control cognitive and behavioral attributes. An increased number of the DA receptor 4 (DRD4) is detected in brain regions primarily involved in cognition. Impairments in executive function have also been reported with depletion in 5HT. A variable number of tandem repeat (VNTR) in the exon 3 of DRD4 and an insertion/deletion polymorphism in the promoter region of 5HT transporter (5HTTLPR) have been found to be associated with different neurobehavioral disorders; however, association of these polymorphisms with DS has never been explored. The present family-based analysis on DS revealed significant over-transmission of a DRD4 VNTR allele which encodes for D4 receptor with average activity. No association was noticed for the 5HTTLPR. We may conclude that these genetic polymorphisms are not contributing to the neuromotor and cognitive dysfunctions observed in DS.

Although genetic, nutritional, and environmental factors have been found to aggravate mental retardation in approximately 1% of individuals, no cause is known till date. In this study, two genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR), C677T (rs#1801133) and A1298C (rs#1801131), have been investigated in idiopathic mental retardation (IMR) subjects. Significantly higher frequency of the C677 allele was observed in IMR (n = 155; chi(2) = 5.5; P = 0.019) and moderate IMR (n = 67; chi(2) = 6.16; P = 0.013) groups as compared to controls (n = 126); for A1298C, no significant difference was noticed. TDT analysis revealed preferential transmission of C677 allele to a small group of mild IMR probands (chi(2) = 5.545; P = 0.018). Higher frequency of CA haplotype was also noticed in IMR cases as compared to controls (chi(2) = 6.28; P = 0.012). We infer from the present investigation that these polymorphisms are not contributing to the aetiology of IMR in this population since both case-control and family-based analysis revealed no significant transmission of the mutated allele.

Trisomy of the 21{st} chromosome leads to an over dosage of several regulatory genes in Down syndrome (DS). Though allelic and genotypic combinations formed between genes are interesting, till date, this particular area has never been explored in DS. In the present investigation four SNPs in two transcription factors, Single minded 2 (SIM2) and V-ets erythroblastosis virus E26 oncogene homolog2 (ETSformula2/formula), located in the 21{st} chromosome were genotyped to understand their role in DS. Genomic DNA of eastern Indian probands with DS (N=132), their parents (N=209) and ethnically matched controls (N=149) was subjected to PCR-based analyses of functionally important SNPs followed by statistical analyses. ETS2 rs461155 showed high heterozygosity in DS. Significantly lower frequency of SIM2 C-G haplotype (rs2073601-rs2073416) was noticed in individuals with DS (P value =0.01669) and their fathers (P value=0.01185). Significantly lower frequency of the A-C-C-G with higher frequency of A-C-A-G haplotypes was also noticed in subjects with DS (P value =0.02089 and 0.00588 respectively). Data obtained indicate that the rs2073601 'A' allele, responsible for nonsynonymous substitution of leucine to methionine, may have some role in DS in this population.

Different components of the folate metabolic cycle are crucial for maintaining integrity of DNA. The present study was aimed at exploring the role of some important constituents of the folate cycle in the etiology of idiopathic intellectual disability (IID). Nuclear families with IID probands (n=226) and ethnically matched controls (n=181) were recruited for micronucleus, karyotype, genetic polymorphism (MTR rs1805087, MTRR rs1801394, and DHFR rs70991108), folate, vitamin B6, vitamin B12, and cysteine analysis. Significant difference in genotype frequencies in IID probands and their parents were observed for rs1805087 (P=0.03, 0.02), rs1801394 (P=0.03, 0.001), and rs70991108 ((P=0.03, 0.02) as compared to controls. IID probands showed significantly higher micronucleus frequency (P=0.01) and decreased vitamin B6 level (P=0.002). A strong correlation between rs1801394 'G' allele and micronucleus was also noticed. From the present investigation, a role of genetic polymorphisms and vitamin B6 levels could be hypothesized in the etiology of IID.

V-ets erythroblastosis virus E26 oncogene homolog2 (ETS2), located at chromosome 21 and overexpressed in Down’s syndrome (DS), has known cancer regulatory functions. Because leukemia is of common occurrence in DS subjects while solid tumors are rare, we have explored the role of ETS2 functional genetic polymorphisms in this differential oncological development. In silico methods were used for identifying deleterious SNPs, tagged SNPs, and linkage disequilibrium followed by genotyping of 14 SNPs in Indo-Caucasoid individuals (N = 668). Significantly different allelic frequencies for rs457705, rs1051420, and rs1051425 were observed in Indian controls (N = 149) compared to other ethnic groups. A heterozygous “T” insertion, between chromosomal contig positions 40195541 and 40195542, was observed in DS subjects and their parents. rs461155 showed significant allelic and genotypic association in breast and oral cancer patients. Significantly higher occurrence of G-C haplotype (rs461155-rs1051425) was also observed in these patients compared to DS and leukemic patients. This is the first report on this type of allelic discrimination pattern of ETS2 under different disease conditions. From the data obtained it may be proposed that allelic discrimination of deleterious SNPs in ETS2 may play a regulatory role in the differential development of malignancy in DS subjects.

Screening for Fragile X syndrome (FRAXA), the most common genetic cause for mental retardation (MR), has mostly been carried out among MR patients. The present study was conducted to find out prevalence of FRAXA amongst children residing in the rural areas of West Bengal.Demographic details including age, sex, nutritional status as well as birth, medical, and developmental histories, were collected amongst rural children (n=38,803) of West Bengal, India, over three years (2004-2007). Based on the records of scholastic backwardness, 179 children were short-listed and examined by a team of experts comprising of child psychiatrist, clinical psychologist, paediatrician and special educator. Blood samples were collected and molecular and cytogenetic studies were performed for identification of CGG repeats and determination of FMR1 gene promoter methylation.Of the selected 179 children, six were diagnosed as Down syndrome, one as cerebral palsy and 140 as non-syndromic MR. These 140 children with MR were grouped as mild (56), moderate (60), and severely (4) retarded based on IQ; children5 yr were grouped as developmental delay (20). FRAXA was not detected in any of these children (frequency being 0% with 0-.02% confidence interval). Prevalence of MR was found to be low (about 4/1000 children). Down syndrome also had a lower frequency (0.15/1000 children).The data obtained in the present study indicated that familial disorders like FRAXA were less frequent in the studied population.