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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.

Zymosan and apoptotic cell containing phagosome maturation in macrophages

Tumor-associated macrophages (TAM) are causally associated with tumorigenesis as well as regulation of antitumor immune responses and have emerged as potential immunotherapeutic targets. Recent evidence suggests TAM phagocytose apoptotic tumor cells within the tumor microenvironment through efferocytosis in an immunologically silent manner, thus maintaining an immunosuppressed microenvironment. The signal transduction pathways coupling efferocytosis and immunosuppression are not well known. Neuropilin-2 (NRP2) is a member of the membrane-associated neuropilin family and has been reported in different immune cells but is poorly characterized. In this study, we show that NRP2 is expressed during macrophage differentiation, is induced by tumor cells, and regulates phagocytosis in macrophages. Furthermore, NRP2 in TAM promoted efferocytosis and facilitated tumor growth. Deletion of NRP2 from TAM impaired the clearance of apoptotic tumor cells and increased secondary necrosis within tumors. This resulted in a break in the immune tolerance and reinitiated antitumor immune responses, characterized by robust infiltration of CD8+ T and natural killer cells. This result suggests NRP2 may act as a molecular mediator that connects efferocytosis and immune suppression. Deletion of NRP2 in TAM downregulated several immunosuppressive and tumor-promoting genes and upregulated immunostimulatory genes in the myeloid compartment. Taken together, our study demonstrates that TAM-derived NRP2 plays a crucial role in tumor promotion through efferocytosis, opening the enticing option for the development of effective immunotherapy targeting TAM.Significance: Neuropilin-2 in macrophages promotes tumor growth by regulating efferocytosis of apoptotic tumor cells and orchestrating immune suppression.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5600/F1.large.jpg. Cancer Res; 78(19); 5600–17. ©2018 AACR.

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies, where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface EGFR, arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat– and FYVE-domain–containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies. Cancer Res; 76(2); 418–28. ©2015 AACR.

Analysis of late endosomal protein PIKfyve

PDF file - 70K

Analysis of early and late endosomal proteins

Maturation of Cathepsin following depletion of NRP2 axis in Du145 and PC3 cells

Late endosomal protein Rab7 also affected following depletion of NRP2 or VEGF-C

PDF file - 1.6MB, Confocal microscopy and western blot results showing inhibition of autophagy and increase in chemotherapy-induced cancer cell death by specific inhibition of VEGF-C/NRP-2 axis

The depletion of NRP2 inhibits Rab switch that lead to an accumulation of immature autophagosome and early endosome

Autophagy_live cell

Depletion of NRP2 axis affect EGFR endocytosis

Overexpression of NRP2 or knockdown of WDFY1 in NRP2 depleted cell recovers the early endosomal structure

Supplementary figure legends

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel, targetable, pathways that contribute to tumor progression of PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC has been only little explored. Here, we show that GD2 is expressed on a small subpopulation of PC cells in a subset of patients, especially in metastatic PC. Variable levels of cell surface GD2 expression are seen in most PC cell lines, and the expression is highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2highcell fraction is enriched upon growth of PC cells as tumorspheres and GD2highfraction is enriched in tumorsphere growth. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2-high CRPC cell models led to marked impairment of theirin vitrooncogenic traits, reduced cancer stem cell (CSC) and epithelial-mesenchymal transition (EMT) marker expression and growth as bone-implanted xenograft tumors. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

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.

The purpose of this study is to investigate the correlation of neuropilin-2 (NRP2) and its ligand, vascular endothelial growth factor-C (VEGF-C), with treatment outcomes and resistance in prostate cancer patients. 166 prostate cancer patient serum samples were obtained from the Integrated Cancer Repository for Cancer Research at the University of Nebraska Medical Center. 11 patients were excluded for having a non-prostate genitourinary cancer or more than one concurrent cancer. Patients were categorized as untreated, primary treatment, remission, and secondary treatment based on when their serum sample was collected. Primary treatment indicated current treatment with first-line medical therapy, remission indicated those without evidence of disease after prostatectomy or first-line medical therapy, and secondary therapy indicated second-line treatment currently being given due to treatment-resistant disease. Other variables collected include age, race, ethnicity, prostate-specific antigen (PSA) measured at the time of diagnosis, PSA measured at the time of serum sample collection, Gleason score, and clinical stage using the American Joint Committee on Cancer (AJCC) tumor, node, metastasis (TNM) system. Finally, VEGF-C levels in patient serum were measured using a commercially available ELISA assay (R&D System, Minneapolis, MN). The final data was analyzed using a linear regression model to compare VEGF-C levels between treatment groups, as well as VEGF-C level correlation with each of the other variables collected. Results: All patients were male with an age range of 47 to 85 years, with a mean age of 65.3 years. Most were white (n=135) and non-Hispanic (n=148). VEGF-C levels ranged from 341 pg/ml to 15,983 pg/ml, with a mean of 3,787 pg/ml. Of the 155 patients, 8 were untreated, 26 were in primary therapy, 66 were in remission, and 52 were in secondary therapy. Results showed no statistically significant correlation between VEGF-C levels and PSA at diagnosis, PSA at serum collection, Gleason score, or clinical stage. The most interesting difference between treatment groups was a higher VEGF-C level in remission (mean 63.48 pg/ml) versus secondary therapy (mean 55.31 pg/ml), although this was not statistically significant (p=0.089). Conclusions: Our results indicated that plasma VEGF-C alone is not a prognostic indicator for prostate cancer. Since our studies using cell culture and animal models of prostate cancer have indicated the role of VEGF-C and the NRP2 axis in promoting metastasis and therapy resistance in prostate cancer, it would be useful to determine both the VEGF-C and NRP2 levels in the plasma of the same patients and study whether their level together would predict the outcome of medical therapy. Our ongoing experiments to determine plasma NRP2 levels in this patient cohort should be able to test this hypothesis. Citation Format: Sarah A. Mullen, Dipanwita Das, Samikshan Dutta, Robin R. High, Lynette M. Smith, Benjamin A. Teply, Kaustubh Datta. Identifying a surrogate biomarker for neuropilin-2 axis in serum of prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2168.

Neuropilins (NRPs) are transmembrane proteins involved in vascular and nervous system development by regulating angiogenesis and axon guidance cues. Several published reports have established their role in tumorigenesis. NRPs are detectable in tumor cells of several cancer types and participate in cancer progression. NRP2 is also expressed in endothelial and immune cells in the tumor microenvironment and promotes functions such as lymphangiogenesis and immune suppression important for cancer progression. In this review, we have taken a comprehensive approach to discussing various aspects of NRP2-signaling in cancer, including its regulation, functional significance in cancer progression, and how we could utilize our current knowledge to advance the studies and target NRP2 to develop effective cancer therapies.

RNA polymerase II-associated factor 1 (PAF1)/pancreatic differentiation 2 (PD2) is a core subunit of the human PAF1 complex (PAF1C) that regulates the RNA polymerase II function during transcriptional elongation. PAF1/PD2 has also been linked to the oncogenesis of pancreatic ductal adenocarcinoma (PDAC). Here, we report that PAF1/PD2 undergoes posttranslational modification (PTM) through SUMOylation, enhancing the radiation resistance of PDAC cells. We identified that PAF1/PD2 is preferentially modified by small ubiquitin-related modifier 1 (SUMO 1), and mutating the residues (K)-150 and 154 by site-directed mutagenesis reduces the SUMOylation. Interestingly, PAF1/PD2 was found to directly interact with the promyelocytic leukemia (PML) protein in response to radiation, and inhibition of PAF1/PD2 SUMOylation at K-150/154 affects its interaction with PML. Our results demonstrate that SUMOylation of PAF1/PD2 increased in the radiated pancreatic cancer cells. Furthermore, inhibition of SUMOylation or PML reduces the cell growth and proliferation of PDAC cells after radiation treatment. These results suggest that SUMOylation of PAF1/PD2 interacts with PTM for PDAC cell survival. Furthermore, abolishing the SUMOylation in PDAC cells enhances the effectiveness of radiotherapy. Overall, our results demonstrate a novel PTM and PAF1/PD2 interaction through SUMOylation, and inhibiting the SUMOylation of PAF1/PD2 enhance the therapeutic efficacy for PDAC.

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.

Upregulation of the non-tyrosine kinase receptor neuropilin-2 (NRP2) is described for several tumor entities and correlates with disease progression. Two membrane-anchored isoforms are transcribed from the NRP2 gene that only differ in their terminal exons to encode transmembrane stretches and short cytoplasmic tails, respectively. While NRP2a was shown to support tumor cell proliferation, NRP2b facilitates tumor cell migration, resulting in enhanced metastatic spreading. A third, soluble NRP2 isoform encoding only aminoterminal protein domains exists as dimeric protein and functions in sequestration of the VEGF-C ligand. By using a next-generation sequencing approach to enrich for NRP2 terminal exons, we identified NRP2-Mo83, a novel NRP2 isoform, from a high expressing tumor cell line, and verified secreted NRP2-Mo83 protein by mass spectrometry in cell supernatants. Preliminary mass spectrometric data give an indication of the presence of soluble NRP2-Mo83 protein in human serum samples.NRP2-Mo83 mRNA is expressed at variable quantities in vitro in a panel of bladder carcinoma cell lines, but is downregulated in a small cohort of macrodissected urothelial carcinoma samples. Transcript abundances of a NRP2 transcript classified as non-coding with high sequence similarity to the NRP2-Mo83 3’ terminus confirm the observed downregulation in the TCGA bladder carcinoma cohort.Preliminary data indicate that recombinant NRP2-Mo83 protein exerts a dose-dependent inhibitory effect on tumor cell growth in vitro in all tested bladder carcinoma cell lines in the presence of serum. However, a fraction of cell lines does not respond to NRP2-Mo83 under serum-starved conditions. Proliferation of human endothelial cells (HUVEC) is inhibited in a dose-dependent manner by recombinant NRP2-Mo83 protein in vitro. In vivo, NRP2-Mo83 reveals an anti-angiogenic effect on the chicken chorioallantoic membrane.Recombinant NRP2-Mo83 is secreted by HEK293 cells. Enrichment of endogenous and purification of recombinant NRP2-Mo83 protein is achieved by exploiting its capacity to bind heparin. In contrast to the described s9NRP2 isoform, NRP2-Mo83 is largely monomeric in solution, as verified by size exclusion chromatography. We identified N and O glycosylation as well as the capacity for polysialation as posttranslational modifications of recombinant NRP2-Mo83 protein, similar to membrane anchored NRP2 isoforms. NanoDSF experiments indicate that calcium ions stabilize NRP2-Mo83. We present NRP2-Mo83 as a novel NRP2 splice isoform, validated its expression at the mRNA and protein level. NRP2-Mo83 appears down-regulated during bladder tumorigenesis, congruent with its function in bladder tumor growth inhibition and its anti-angiogenic properties in vitro and in vivo. The identification of NRP2-Mo83 in human serum may suggest a suppressive function even under non-pathological conditions. Citation Format: Thomas Mayr, Paul L. Dix, Sayed-Mohammad Hasheminasab, Chuanpit Hampel, Marc Sylvester, Niels Schneberger, Sarah Foerster, Gregor Hagelueken, Samikshan Dutta, Glen Kristiansen, Regine Schneider-Stock, Kaustubh Datta, Michael H. Muders. Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity [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 831.

Background: Prolonged ablation of androgen receptor pathway in prostate cancer leads to the emergence of therapy resistant neuroendocrine-like (NE-like) aggressive lineage. This NE development is associated with chromatin reprogramming underlying epigenetic alterations and changes in transcriptional network. Clinically, NE development follows a poorer progression with multifaceted therapeutic challenges. Current lack of effective understanding of this NE trans-differentiation process limits the stratification of therapeutic window with further worsened patient management. The following study aims to identify the sequential molecular players responsible for therapy resistant NE like development. Objective: The primary objective of this study is to understand how chromatin modifications along with epigenetic regulation dictates the establishment of NE trans-differentiation process in Prostate Cancer in response to Androgen Receptor blockade therapy. Methods: We generated various NE-like cell lines either by genetic modification or therapeutic selection. We performed RNA Seq, ATAC Seq and acetylated Histone (H3K18 and H3K27) ChIP Seq in our developed NE and adenocarcinoma cell lines. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during adenocarcinoma to NE transformation. Overlapping RNA seq results further suggested newly transcribed neuronal genes with higher degrees of promoter accessibility. Transcription Factor scanning analysis among newly active gene promoters revealed preferential binding of Pax5, whose expression has been validated to be selectively occurring in NE lineage unlike adenocarcinoma. Further analysis of Pax5 promoter site suggested that changes in 5-hydroxymethylation pattern during adenocarcinoma to NE transformation guided the recruitment of PBX1 at Pax5 promoter. Validating NE patient expression in silico supported that PBX1/Pax5 expressions are selective in NE lineage development. Conclusions: Our study concludes that specific chromatin alterations guide the recruitment of PBX1 to upregulate Pax5 to maintain a therapy resistant neuronal surrounding in NE-like prostate cancer development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Chromatin modifications guide Pax5 dependent gene expression in NE like prostate cancer [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 LB037.

Background & Significance: Prostate cancer (PCa) is the second leading cause of cancer deaths (~34,000 in 2021 (ACS)) in American men. Castration resistance and resistance to the next-gen androgen receptor (AR) targeted drugs are major challenges. Castration resistance involves multiple mechanisms, including androgen-independent signaling by androgen receptor (AR)- or its variants, and lineage plasticity (LP) with AR-indifferent neuroendocrine (NE) differentiation. Identifying new vulnerabilities across these multitude mechanisms could provide new therapeutic avenues against castration-resistant PCa (CRPC). The cell-surface di-ganglioside GD2 is overexpressed in neural crest cell tumors such as neuroblastoma & melanoma and chimeric (Dinutuximab) or humanized (Naxitamab) anti-GD2 antibodies are now FDA-approved for high-risk neuroblastoma therapy. GD2 expression is reported in other cancers such as breast cancer and glioma and is linked to cancer stem cell behavior. While limited prior studies have detected GD2 expression in PCa cell lines or tumor tissues, nothing is known about the functional role of GD2 in PCa. Objectives: We hypothesized that GD2 overexpression in PCa could play a pro-tumorigenic role and that linkage of GD2 overexpression with CRPC progression may reveal the potential of targeting GD2 for CRPC therapy. Study Design & Results: Immunohistochemical analysis of PCa patient and patient-derived xenograft tissue microarrays (TMAs) revealed GD2 expression in a subset of tumor cells. Fluorescence-activated cell sorter analysis of PCa cell lines showed strong constitutive GD2 expression on murine CRPC cell line RM-1 (derived from mutant Ras and c-Myc overexpressing prostatic epithelial cells) and human PCa line 22Rv1 (overexpresses wild-type AR and ARv7 splice variant). GD2 expression was induced de novo upon induction of lineage plasticity in GD2-negative LNCaP C4-2 prostate adenocarcinoma cell line by shRNA knockdown (KD) of RB1 or TP53. High GD2 expression was also induced when C4-2B cells were made enzalutamide resistant (C4-2BER). Induction of GD2 expression correlated with increased expression of rate-limiting GD2 biosynthetic pathway enzyme GD3 synthase (GD3S). CRISPR-Cas9 mediated stable GD3S knockout (KO) in the RM1 cell line led to the loss of GD2 expression. The GD3S-KO RM1 cells exhibited reduced proliferation, migration, invasion, and tumor sphere forming ability compared to the control cells. Intratibial injections in castrated male C57BL/6 mice showed a significant reduction in tumor development by GD3S KO RM1 cells compared to control cells. Conclusions: Our studies demonstrate that GD2 is expressed in a subset of prostate cancers. Cell line-based studies show that GD2 expression promotes pro-tumorigenic traits. Future studies will assess the biological roles of GD2 in PCa and the potential of targeting GD2+ CRPC with antibody-based approved therapeutic agents. Citation Format: Aaqib M. Bhat, Bhopal C. Mohapatra, Insha Mushtaq, Sukanya Chakraborty, Samikshan Dutta, Sameer Mirza, Matthew D. Storck, Subodh M. Lele, Ming-Fong Lin, Bruce J. Trock, Karen S. Sfanos, Colm Morrissey, Eva Corey, Jonathan Melamed, Leah Cook, Kaustubh Datta, Jane Meza, Jawed Siddiqui, Surinder K. Batra, Vimla Band, Hamid Band. Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer [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 2411.

Prostate cancer (PCa) is the leading cause of cancer death in men. With more therapeutic modalities available, the overall survival in PCa has increased significantly in recent years. Patients with relapses after advanced secondgeneration anti-androgen therapy however, often show poor disease prognosis. This group of patients often die from cancer-related complicacies. Multiple approaches have been taken to understand disease recurrence and to correlate the gene expression profile. In one such study, an 11-gene signature was identified to be associated with PCa recurrence and poor survival. Amongst them, a specific deubiquitinase called ubiquitin-specific peptidase 22 (USP22) was selectively and progressively overexpressed with PCa progression. Subsequently, it was shown to regulate androgen receptors and Myc, the two most important regulators of PCa progression. Furthermore, USP22 has been shown to be associated with the development of therapy resistant PCa. Inhibiting USP22 was also found to be therapeutically advantageous, especially in clinically challenging and advanced PCa. This review provides an update of USP22 related functions and challenges associated with PCa research and explains why targeting this axis is beneficial for PCa relapse cases.

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Background: Prostate cancer (PCa) patient management is becoming increasingly challenging owing to the emergence of advanced therapy-resistant PCa. Published reports from our laboratory demonstrated that neuropilin-2 (NRP2), a non-tyrosine kinase transmembrane receptor, is associated with therapy resistance in aggressive PCa. Such advanced PCa is often associated with diverse genetic changes that complicate the therapeutic interventions. One example is the loss of tumor suppressor gene TP53 along with RB1 to develop therapy resistant neuroendocrine (NE)-like PCa that evades the standard androgen depletion therapy (ADT) and is resistant to first-line chemotherapy. Our results indicate that loss of TP53 and RB1 is associated with significantly increased expression of NRP2. Significance of the problem: Switch to NE-like lineage has been found to be associated with resistance to androgen receptor (AR) targeted therapy as well as chemotherapy. Patients with NE-like PCa show poor prognosis owing to the heterogeneous feature of the tumors and unavailability of effective therapy against this type of cancer. Therefore, identification of potential molecular target(s) is necessary to combat this disease. Objective: The objective of this study is to comprehend the underlying molecular mechanism/s of increased NRP2 expression in PCa, and whether targeting NRP2 would be effective against therapy resistant NE-like PCa, especially in combination with first-line chemotherapeutic agents. Study design: Current study was performed to test the NRP2 status in NE-like PCa cells and to understand if NRP2 is associated with the therapy resistance in this type of aggressive cancer. Moreover, efficacy of NRP2 depletion to sensitize the cancer cells to the chemotherapeutic agents used clinically was evaluated. A stable cell line model of NE-like PCa was developed and characterized by knocking down TP53 and RB1 genes in castration resistant prostate cancer (CRPC) cells. The resistance of the cells against the clinically used chemotherapy was investigated. In addition, efficacy of the chemotherapeutic agents following NRP2 knockdown was evaluated in in-vitro and in-vivo models. For in-vivo studies, PCa bone metastatic mouse model was developed in our laboratory and the effect of the depletion of NRP2 in combination with chemotherapeutic agent was evaluated. Results: NRP2 was significantly expressed in the NE-like PCa cells and knockdown of NRP2 sensitized the originally resistant NE-like cells to chemotherapeutic drugs. Tumor burden was also reduced in the bone metastatic mouse model upon NRP2 depletion when given in combination with chemotherapy. Conclusion: Therefore, targeting NRP2 can be an effective therapeutic strategy to combat highly aggressive and therapy resistant NE-like PCa. Citation Format: Ridwan Islam, Navatha Polavaram, Zhengdong Hong, Sreyashi Bhattacharya, Sanika Bodas, Benjamin Teply, Michael Muders, Kaustubh Datta, Samikshan Dutta. Neuropilin-2, a potential target against neuroendocrine like therapy resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1422.

Background:Failure of castration resistant prostate cancer patients to Androgen Receptor Signaling Inhibitor (ARSI) poses a major challenge in terms of therapeutic development. The heterogeneous nature of prostate cancer makes it difficult to stratify the treatment modalities. A multitude of variable pathological and genetic alterations induced by systemic treatment adds further to the therapeutic complexity. Upon continued exposure to enzalutamide (ARSI), nearly 15-20% of castration resistant patients advance towards a highly aggressive neuroendocrine like phenotype with concomitant functional loss of RB1 and TP53 and persistent AR expression in some. Elucidation of the underlying mechanism of resistance among this aggressive subset of patients is of utmost importance to clinically stratify the treatment options and target these advanced neuroendocrine like patients, who currently lack specific treatment approaches. The following study aims to identify the molecular signature responsible for driving this enzalutamide resistant neuroendocrine like development. Objective:The primary objective of the study is to identify the molecular mechanism behind ARSI induced neuroendocrine like Prostate cancer development. Methods:We have developed two cell line model systems. C4-2 has been genetically modified to develop a simultaneous genetic loss of RB1 and TP53 (DKD). C4-2B has been kept on continued exposure to Enzalutamide to develop Enzalutamide Resistant (C4-2BER). C4-2BER maintains AR expression while DKD loses AR expression, resembling different stages in neuroendocrine like development. Using Western Blot and RT PCR analysis the neuroendocrine like background of these resistant cell line models has been characterized. We performed an RNA-Seq with C4-2B and its resistant model C4-2BER and further downstream analysis through gene ontology and GSEA were performed. Results:Model system characterization study revealed neuroendocrine like characteristics and gene expression. Genetic analysis revealed an upregulation of multiple genes in C4-2BER in comparison to its parental C4-2B, which is comparable to patient dataset available online. GSEA analysis revealed several neuronal biology related pathways to be prominently upregulated among C4-2BER. Further analysis revealed that expression of WT1, which binds near most of these upregulated genes, is downregulated in C4-2BER. Conclusions:The downregulation of WT1 is noted during progression from CRPC towards enzalutamide resistant neuroendocrine development in C4-2BER, which is in turn accompanied with various epigenetic changes. Understanding such pattern of changes and identifying the underlying molecular signature would help elucidate the targets behind treatment induced neuroendocrine characteristics from a therapeutic point of view. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Navatha Polavaram, Kaustubh Datta, Samikshan Dutta. Second generation androgen receptor signaling inhibitor resistant neuroendocrine like prostate cancer: A mechanistic study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1326.

INTRODUCTION:Patients with advanced prostate cancer often report resistance to standard ARSI (Androgen Receptor Signaling Inhibitor) therapy. One reason for such therapy resistance is the development of splice variants of Androgen Receptor (AR). Additionally, studies have reported AR-independent signaling axis to play an important role in conferring therapy resistance in these cancers. The purpose of this study is to explore the underlying molecular mechanisms of resistance and develop an alternate therapeutic strategy for such patients.OBJECTIVES: In this study we investigated the role of Neuropilin-2 (NRP2), a receptor protein often upregulated in advanced prostate cancer, in imparting therapy-resistance to ARSI treatment (like enzalutamide). The final objective of this study is to develop a treatment strategy which targets NRP2 along with Enzalutamide treatment to increase treatment efficacy in therapy-resistant prostate cancer. METHODS: To establish the expression pattern of NRP2 in advanced prostate cancer, we analyzed a dataset of 396 patients with advanced prostate cancer. Immunohistochemistry was performed on tissue samples to observe NRP2 localization within the cancer cells. NRP2 was knocked down using two independent siRNAs in various prostate cancer cell lines to determine the effect of NRP2 inhibition on cancer growth and survival. The knockdown was validated by Western Blot and RT-PCR. The efficacy of NRP2 inhibition was quantitated by measuring cell death using YO-PRO-I and PI assay for cellular apoptosis. In vitro tumorigenicity of cells after NRP2 inhibition in combination with enzalutamide was assessed using soft agar colony formation assay. RESULTS:Our patient datasets showed a significant upregulation of NRP2, as well as poor cancer-specific survival, in advanced prostate cancer patients with higher Gleason scores. Immunohistochemistry analyses revealed a markedly high expression of NRP2 in the nucleus of cancer cells. We found that NRP2 nuclear translocation enabled transcription of novel oncogenes by interacting with AR. Knockdown of NRP2 was shown to bring about increased cell death in vitro. Inhibiting NRP2 was also seen to reduce AR-regulated oncogenesis. Cancer cell tumorigenicity was significantly reduced when NRP2 was inhibited in combination with enzalutamide, as evidenced by the decreased number of colonies in our colony formation assay. CONCLUSION:Our results indicate that inhibiting NRP2 in combination with enzalutamide sensitizes the cells to ARSI treatment. Thus, NRP2 inhibition along with enzalutamide can be further explored as a treatment option for patients with treatment-refractory prostate cancer. Citation Format: Sanika Bodas, Ridwan Islam, Navatha Polavaram, Sreyashi Bhattacharya, Michael Muders, Samikshan Dutta, Benjamin A. Teply, Kaustubh Datta. Inhibiting Neuropilin-2 overcomes resistance to enzalutamide in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1409.

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.

Tumor-associated macrophages (TAM) are causally associated with tumorigenesis as well as regulation of antitumor immune responses and have emerged as potential immunotherapeutic targets. Recent evidence suggests TAM phagocytose apoptotic tumor cells within the tumor microenvironment through efferocytosis in an immunologically silent manner, thus maintaining an immunosuppressed microenvironment. The signal transduction pathways coupling efferocytosis and immunosuppression are not well known. Neuropilin-2 (NRP2) is a member of the membrane-associated neuropilin family and has been reported in different immune cells but is poorly characterized. In this study, we show that NRP2 is expressed during macrophage differentiation, is induced by tumor cells, and regulates phagocytosis in macrophages. Furthermore, NRP2 in TAM promoted efferocytosis and facilitated tumor growth. Deletion of NRP2 from TAM impaired the clearance of apoptotic tumor cells and increased secondary necrosis within tumors. This resulted in a break in the immune tolerance and reinitiated antitumor immune responses, characterized by robust infiltration of CD8+ T and natural killer cells. This result suggests NRP2 may act as a molecular mediator that connects efferocytosis and immune suppression. Deletion of NRP2 in TAM downregulated several immunosuppressive and tumor-promoting genes and upregulated immunostimulatory genes in the myeloid compartment. Taken together, our study demonstrates that TAM-derived NRP2 plays a crucial role in tumor promotion through efferocytosis, opening the enticing option for the development of effective immunotherapy targeting TAM. Significance: Neuropilin-2 in macrophages promotes tumor growth by regulating efferocytosis of apoptotic tumor cells and orchestrating immune suppression. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5600/F1.large.jpg. Cancer Res; 78(19); 5600–17. ©2018 AACR.

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.

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the γ-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

Background: RNA polymerase associated factor 1 (PAF1)/Pancreatic differentiation 2 (PD2) is one of the core subunit of the human PAF1 complex (PAF1C), which regulates various cellular functions such as transcriptional elongation and histone modification. We have previously demonstrated its unique role in oncogenesis and stem cell maintenance. Studies have demonstrated that PAF1/PD2 gene yields a protein of 59.9 Kda (531 amino acids), however it has been found that it always gives a band at 80 Kda. Further, previous studies suggests that the 60 Kda protein represents the precursor, which rapidly process into an 80 Kda mature protein. SUMOylation is a process of reversible posttranslational modification that adds a small ubiquitin-related modifier (SUMO)-1 protein to the target protein. SUMOylation plays various molecular biology function such as transcriptional regulation, protein-protein interaction, and DNA damage repair and in cell cycle. DNA damage happen due to several physiological processes, but can also be caused by genotoxic agents. Promyelocytic Leukemia (PML) is protein that forms nuclear bodies and may be modified by SUMO1 and act as a DNA-damage sensor. Hypothesis: PAF1/PD2 is interacting with SUMO-1 and PML and, thus, plays an important function in providing gemcitabine resistance to pancreatic cancer cells Methods: SW1990, F9 (mouse embryonic cells) and CD18/HPAF cells were treated with 2-D08, a potent SUMOylation inhibitor for 24 hours and with siRNA against SUMO1 for 48 hours followed by protein isolation and western blotting. Immunoprecipitation and immunofluorescence were done to show the interaction between PAF1/PD2 and SUMO1 and with PML. To study the effect of PAF1/PD2 on gemcitabine resistance, SW1990 and Capan-1 cells were treated with different concentration of gemcitabine and then the expression of PAF1/PD2 along with SUMO1 was checked through immunoblotting and confocal imaging. Results: Results shows that inhibiting SUMOylation with both 2D08 and siRNA resulted in a decrease expression of PAF1/PD2 80 Kda protein. Immunoprecipitation and immunofluorescence analysis showed that endogenous PAF1/PD2 interacts with SUMO1. This finding was further verified using ectopically overexpressed Flag- tagged PAF1/PD2 and HA-tagged SUMO1, which showed a physical interaction between PAF1/PD2 and SUMO1. Interestingly, we observed that gemcitabine treatment significantly increased the SUMOylated status of PAF1/PD2 in pancreatic cancer cells. Further our results proved that SUMOylated PAF1/PD2 form nuclear bodies along with PML in pancreatic cancer cells. Conclusions: Our observation suggest that PAF1/PD2 undergoes SUMOylation and covalently interacts with SUMO1. Treatment with gemcitabine results in enhanced expression of PAF1/PD2 and increased co-localization with SUMO1 and PML, indicates a role of SUMOylated PAF1/PD2 in gemcitabine resistance. Citation Format: Sanchita Rauth, Saswati Karmakar, Ashu Shah, Rama Krishna Nimmakayala, Rakesh Bhatia, Sakthivel Muniyan, Sushil Kumar, Samikshan Dutta, Kaustubh Datta, Surinder K. Batra, Moorthy Palanimuthu Ponnusamy. Role of post translational modification of PAF1/PD2 in gemcitabine resistance of pancreatic 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 4438.

A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1, that have previously been suggested to participate in autophagy and vesicular trafficking. Upregulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C/NRP-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies. Cancer Res; 73(1); 160–71. ©2012 AACR.

Neuropilins (NRPs) are cell surface glycoproteins that often act as co-receptors for plexins and VEGF family receptors. Neuropilin-2 (NRP2), a family member of NRPs, was shown to regulate autophagy and endocytic trafficking in cancer cells, a function distinctly different from its role as a co-receptor. WD Repeat and FYVE domain containing 1 (WDFY1)–protein acts downstream of NRP2 for this function. Our results indicated that NRP2 maintains an optimum concentration of WDFY1 by negatively regulating its expression. Since increased expression of WDFY1 reduces the endocytic activity, maintenance of WDFY1 level is crucial in metastatic cancer cells to sustain high endocytic activity, essential for promotion of oncogenic activation and cancer cell survival. Here, we have delineated the underlying molecular mechanism of WDFY1 synthesis by NRP2. Our results indicated that NRP2 inhibits WDFY1 transcription by preventing the nuclear localization of a transcription factor, Fetal ALZ50-reactive clone 1 (FAC1). Our finding is novel as transcriptional regulation of a gene by NRP2 axis has not been reported previously. Regulation of WDFY1 transcription by NRP2 axis is a critical event in maintaining metastatic phenotype in cancer cells. Thus, inhibiting NRP2 or hyper-activating WDFY1 can be an effective strategy to induce cell death in metastatic cancer.

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.

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies, where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface EGFR, arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat– and FYVE-domain–containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies. Cancer Res; 76(2); 418–28. ©2015 AACR.

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.

Septins, a conserved family of GTP-binding proteins with a conserved role in cytokinesis, are present in eukaryotes ranging from yeast to mammals. Septins are also highly expressed in neurons, which are post-mitotic cells. Septin6 (SEPT6) forms SEPT2/6/7 complexes in vivo. In this study, we produced a very specific SEPT6 antibody. Immunocytochemisty (ICC) of dissociated hippocampal cultures revealed that SEPT6 was highly expressed in neurons. Developmentally, the expression of SEPT6 was very low until stage 3 (axonal outgrowth). Significant expression of SEPT6 began at stage 4 (outgrowth of dendrites). At this stage, SEPT6 clusters were positioned at the branch points of developing dendrites. In maturing and mature neurons (stage 5), SEPT6 clusters were positioned at the base of filopodia and spines, and pre-synaptic boutons. Detergent extraction experiments also indicated that SEPT6 is not a post-synaptic density (PSD) protein. Throughout morphologic development of neurons, SEPT6 always formed tiny rings (external diameter, ∼0.5 μm), which appear to be clusters at low magnification. When a Sept6 RNAi vector was introduced at the early developmental stage (DIV 2), a significant reduction in dendritic length and branch number was evident. Taken together, our results indicate that SEPT6 begins to be expressed at the stage of dendritic outgrowth and regulates the cytoarchitecture.