An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer.

Elevated serine peptidase inhibitor, Kazal type 1 (SPINK1) levels in ∼10%-25% of prostate cancer (PCa) patients associate with aggressive phenotype, for which there are limited treatment choices and dismal clinical outcomes. Using an integrative proteomics approach involving label-free phosphoproteome and proteome profiling, we delineated the downstream signaling pathways involved in SPINK1-mediated tumorigenesis and identified tyrosine kinase KIT as highly enriched. Furthermore, high to moderate levels of KIT expression were detected in ∼85% of SPINK1-positive PCa specimens. We show KIT signaling orchestrates SPINK1-mediated oncogenesis, and treatment with KIT inhibitor reduces tumor growth and metastases in preclinical mice models. Mechanistically, KIT signaling modulates WNT/ß-catenin pathway and confers stemness-related features in PCa. Notably, inhibiting KIT signaling led to restoration of AR/REST levels, forming a feedback loop enabling SPINK1 repression. Overall, we uncover the role of KIT signaling downstream of SPINK1 in maintaining lineage plasticity and provide distinct treatment modalities for advanced-stage SPINK1-positive patients.

Targeting MALAT1 Augments Sensitivity to PARP Inhibition by Impairing Homologous Recombination in Prostate Cancer.

PARP inhibitors (PARPi) have emerged as a promising targeted therapeutic intervention for metastatic castrate-resistant prostate cancer (mCRPC). However, the clinical utility of PARPi is limited to a subset of patients who harbor aberrations in the genes associated with the homologous recombination (HR) pathway. Here, we report that targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an oncogenic long noncoding RNA (lncRNA), contrives a BRCAness-like phenotype, and augments sensitivity to PARPi. Mechanistically, we show that MALAT1 silencing reprograms the homologous recombination (HR) transcriptome and makes prostate cancer cells more vulnerable to PARPi. Particularly, coinhibition of MALAT1 and PARP1 exhibits a decline in clonogenic survival, delays resolution of γH2AX foci, and reduces tumor burden in mice xenograft model. Moreover, we show that miR-421, a tumor suppressor miRNA, negatively regulates the expression of HR genes, while in aggressive prostate cancer cases, miR-421 is sequestered by MALAT1, leading to increased expression of HR genes. Conclusively, our findings suggest that MALAT1 ablation confers sensitivity to PARPi, thus highlighting an alternative therapeutic strategy for patients with castration-resistant prostate cancer (CRPC), irrespective of the alterations in HR genes. SIGNIFICANCE: PARPi are clinically approved for patients with metastatic CRPC carrying mutations in HR genes, but are ineffective for HR-proficient prostate cancer. Herein, we show that oncogenic lncRNA, MALAT1 is frequently overexpressed in advanced stage prostate cancer and plays a crucial role in maintaining genomic integrity. Importantly, we propose a novel therapeutic strategy that emphasizes MALAT1 inhibition, leading to HR dysfunction in both HR-deficient and -proficient prostate cancer, consequently augmenting their susceptibility to PARPi.

Human ERG oncoprotein represses a Drosophila LIM domain binding protein-coding gene Chip.

Human ETS Related Gene, ERG, a master transcription factor, turns oncogenic upon its out-of-context activation in diverse developmental lineages. However, the mechanism underlying its lineage-specific activation of Notch (N), Wnt, or EZH2-three well-characterized oncogenic targets of ERG-remains elusive. We reasoned that deep homology in genetic tool kits might help uncover such elusive cancer mechanisms in Drosophila. By heterologous gain of human ERG in Drosophila, here we reveal Chip, which codes for a transcriptional coactivator, LIM-domain-binding (LDB) protein, as its novel target. ERG represses Drosophila Chip via its direct binding and, indirectly, via E(z)-mediated silencing of its promoter. Downregulation of Chip disrupts LIM-HD complex formed between Chip and Tailup (Tup)-a LIM-HD transcription factor-in the developing notum. A consequent activation of N-driven Wg signaling leads to notum-to-wing transdetermination. These fallouts of ERG gain are arrested upon a simultaneous gain of Chip, sequestration of Wg ligand, and, alternatively, loss of N signaling or E(z) activity. Finally, we show that the human LDB1, a homolog of Drosophila Chip, is repressed in ERG-positive prostate cancer cells. Besides identifying an elusive target of human ERG, our study unravels an underpinning of its lineage-specific carcinogenesis.

Transcription networks rewire gene repertoire to coordinate cellular reprograming in prostate cancer.

Transcription factors (TFs) represent the most commonly deregulated DNA-binding class of proteins associated with multiple human cancers. They can act as transcriptional activators or repressors that rewire the cistrome, resulting in cellular reprogramming during cancer progression. Deregulation of TFs is associated with the onset and maintenance of various cancer types including prostate cancer. An emerging subset of TFs has been implicated in the regulation of multiple cancer hallmarks during tumorigenesis. Here, we discuss the role of key TFs which modulate transcriptional cicuitries involved in the development and progression of prostate cancer. We further highlight the role of TFs associated with key cancer hallmarks, including, chromatin remodeling, genome instability, DNA repair, invasion, and metastasis. We also discuss the pluripotent function of TFs in conferring lineage plasticity, that aids in disease progression to neuroendocrine prostate cancer. At the end, we summarize the current understanding and approaches employed for the therapeutic targeting of TFs and their cofactors in the clinical setups to prevent disease progression.

Red-emitting polyaniline-based nanoparticle probe for pH-sensitive fluorescence imaging.

Fluorescent probes based on semiconducting polymer nanoparticles (NPs) such as polyaniline (PANI) usually require external fluorophore doping to provide fluorescence function. Direct use of PANI-based NPs for bioimaging applications has been limited by PANI's weak blue fluorescence and aggregation-induced quenching in physiological medium. In this report, we developed a facile solid-state synthesis method to produce fluorescent polyaniline nanoparticles (FPNs) that are not only water-soluble but also exhibit high intensity and pH-sensitive red fluorescence. The FPNs showed high photoluminescence quantum yield (PLQY) of 19.3 % at physiological pH, which makes FPNs ideal for application as fluorescent nanoprobes in bioimaging. Moreover, we performed an in-depth study of photoluminescence dependence on pH and the phenomena of exciton-polaron quenching at low pH was highlighted. We also found that the ratio of emission intensity at 600 nm and 650 nm increased from 0.04 to 1.65 as pH was raised from 2.6 to 11.8, which could find its application in ratiometric pH sensing. FPNs exhibited excellent biocompatibility with >85 % cell viability for fibroblasts NIH/3 T3 and prostate cancer 22RV1 cells even at concentrations as high as 1000 µg/mL. In addition, fluorescence microscopy demonstrated concentration-dependent red fluorescence in the cytoplasm owing to the cellular uptake of FPNs in prostate cancer cells.

Untargeted Metabolomics Showed Accumulation of One-Carbon Metabolites to Facilitate DNA Methylation during Extracellular Matrix Detachment of Cancer Cells.

Tumor cells detached from the extracellular matrix (ECM) undergo anoikis resistance and metabolic reprogramming to facilitate cancer cell survival and promote metastasis. During ECM detachment, cancer cells utilize genomic methylation to regulate transcriptional events. One-carbon (1C) metabolism is a well-known contributor of SAM, a global substrate for methylation reactions, especially DNA methylation. DNA methylation-mediated repression of NK cell ligands MICA and MICB during ECM detachment has been overlooked. In the current work, we quantitated the impact of ECM detachment on one-carbon metabolites, expression of 1C regulatory pathway genes, and total methylation levels. Our results showed that ECM detachment promotes the accumulation of one-carbon metabolites and induces regulatory pathway genes and total DNA methylation. Furthermore, we measured the expression of well-known targets of DNA methylation in NK cell ligands in cancer cells, namely, MICA/B, during ECM detachment and observed low expression compared to ECM-attached cancer cells. Finally, we treated the ECM-detached cancer cells with vitamin C (a global methylation inhibitor) and observed a reduction in the promoter methylation of NK cell ligands, resulting in MICA/B re-expression. Treatment with vitamin C was also found to reduce global DNA methylation levels in ECM-detached cancer cells.

The Histone H3K27me3 Demethylases KDM6A/B Resist Anoikis and Transcriptionally Regulate Stemness-Related Genes.

Epithelial cancer cells that lose attachment from the extracellular matrix (ECM) to seed in a distant organ often undergo anoikis's specialized form of apoptosis. Recently, KDM3A (H3K9 demethylase) has been identified as a critical effector of anoikis in cancer cells. However, whether other histone demethylases are involved in promoting or resisting anoikis remains elusive. We screened the major histone demethylases and found that both H3K27 histone demethylases, namely, KDM6A/B were highly expressed during ECM detachment. Inhibition of the KDM6A/B activity by using a specific inhibitor results in reduced sphere formation capacity and increased apoptosis. Knockout of KDM6B leads to the loss of stem cell properties in solitary cells. Furthermore, we found that KDM6B maintains stemness by transcriptionally regulating the expression of stemness genes SOX2, SOX9, and CD44 in detached cells. KDM6B occupies the promoter region of both SOX2 and CD44 to regulate their expression epigenetically. We also noticed an increased occupancy of the HIF1α promoter by KDM6B, suggesting its regulatory role in maintaining hypoxia in detached cancer cells. This observation was further strengthened as we found a significant positive association in the expression of both KDM6B and HIF1α in various cancer types. Overall, our results reveal a novel transcriptional program that regulates resistance against anoikis and maintains stemness-like properties.

Nuclear magnetic resonance spectroscopy reveals dysregulation of monounsaturated fatty acid metabolism upon SPINK1 attenuation in colorectal cancer.

Metabolic reprogramming, a key hallmark of cancer, plays a pivotal role in fulfilling the accelerated biological demands of tumor cells. Such metabolic changes trigger the production of several proinflammatory factors, thereby inciting cancer development and its progression. Serine protease inhibitor Kazal Type 1 (SPINK1), well known for its oncogenic role and its upregulation via acute-phase reactions, is highly expressed in multiple cancers including colorectal cancer (CRC). Here, we show accumulation of lipid droplets in CRC cells stained with Oil Red O upon SPINK1 silencing. Furthermore, NMR spectroscopy analysis revealed an accretion of monounsaturated fatty acids (MUFAs) and phosphatidylcholine in these CRC cells, while the levels of polyunsaturated fatty acids remained unaltered. This alteration indicates the presence of MUFAs with the triglycerides in the lipid droplets as observed in SPINK1-silenced CRC cells. Considering the role of MUFAs in the anti-inflammatory response, our data hint that suppression of SPINK1 in CRC leads to activation of an anti-inflammatory signaling milieu. Conclusively, our study uncovers a connection between lipid metabolism and SPINK1-mediated CRC progression, hence paving the way for further exploration and better prognosis of SPINK1-positive CRC patients.

Epigenetic reprogramming during prostate cancer progression: A perspective from development.

Conrad Waddington's theory of epigenetic landscape epitomize the process of cell fate and cellular decision-making during development. Wherein the epigenetic code maintains patterns of gene expression in pluripotent and differentiated cellular states during embryonic development and differentiation. Over the years disruption or reprogramming of the epigenetic landscape has been extensively studied in the course of cancer progression. Cellular dedifferentiation being a key hallmark of cancer allow us to take cues from the biological processes involving epigenetic reprogramming in development such as the cellular differentiation and morphogenesis. Here, we discuss the role of epigenetic landscape and its modifiers in cell-fate determination, differentiation and prostate cancer progression. Lately, the emergence of RNA-modifications has also furthered our understanding of epigenetics in cancer. The overview of the epigenetic code regulating androgen signalling, and progression to aggressive neuroendocrine stage of PCa reinforces its gene regulatory functions during the development of prostate gland as well as cancer progression. Additionally, we also highlight the clinical implications of cancer cell epigenome, and discuss the recent advancements in the therapeutic strategies targeting the advanced stage disease.

Transcriptional network involving ERG and AR orchestrates Distal-less homeobox-1 mediated prostate cancer progression.

Distal-less homeobox-1 (DLX1) is a well-established non-invasive biomarker for prostate cancer (PCa) diagnosis, however, its mechanistic underpinnings in disease pathobiology are not known. Here, we reveal the oncogenic role of DLX1 and show that abrogating its function leads to reduced tumorigenesis and metastases. We observed that ~60% of advanced-stage and metastatic patients display higher DLX1 levels. Moreover, ~96% of TMPRSS2-ERG fusion-positive and ~70% of androgen receptor (AR)-positive patients show elevated DLX1, associated with aggressive disease and poor survival. Mechanistically, ERG coordinates with enhancer-bound AR and FOXA1 to drive transcriptional upregulation of DLX1 in ERG-positive background. However, in ERG-negative context, AR/AR-V7 and FOXA1 suffice to upregulate DLX1. Notably, inhibiting ERG/AR-mediated DLX1 transcription using BET inhibitor (BETi) or/and anti-androgen drugs reduce its expression and downstream oncogenic effects. Conclusively, this study establishes DLX1 as a direct-target of ERG/AR with an oncogenic role and demonstrates the clinical significance of BETi and anti-androgens for DLX1-positive patients.

Nutritive vitamins as epidrugs.

Epigenetic modifications play an important role in disease pathogenesis and therefore are a focus of intense investigation. Epigenetic changes include DNA, RNA, and histone modifications along with expression of non-coding RNAs. Various factors such as environment, diet, and lifestyle can influence the epigenome. Dietary nutrients like vitamins can regulate both physiological and pathological processes through their direct impact on epigenome. Vitamin A acts as a major regulator of above-mentioned epigenetic mechanisms. B group vitamins including biotin, niacin, and pantothenic acid also participate in modulation of various epigenome. Further, vitamin C has shown to modulate both DNA methylation and histone modifications while few reports have also supported its role in miRNA-mediated pathways. Similarly, vitamin D also influences various epigenetic modifications of both DNA and histone by controlling the regulatory mechanisms. Despite the information that vitamins can modulate the epigenome, the detailed mechanisms of vitamin-mediated epigenetic regulations have not been explored fully and hence further detailed studies are required to decipher their role at epigenome level in both normal and disease pathogenesis. The current review summarizes the available literature on the role of vitamins as epigenetic modifier and highlights the key evidences for developing vitamins as potential epidrugs.

Computational Design of BH3-Mimetic Peptide Inhibitors That Can Bind Specifically to Mcl-1 or Bcl-XL: Role of Non-Hot Spot Residues.

Interactions between pro- and anti-apoptotic Bcl-2 proteins decide the fate of the cell. The BH3 domain of pro-apoptotic Bcl-2 proteins interacts with the exposed hydrophobic groove of their anti-apoptotic counterparts. Through their design and development, BH3 mimetics that target the hydrophobic groove of specific anti-apoptotic Bcl-2 proteins have the potential to become anticancer drugs. We have developed a novel computational method for designing sequences with BH3 domain features that can bind specifically to anti-apoptotic Mcl-1 or Bcl-XL. In this method, we retained the four highly conserved hydrophobic and aspartic residues of wild-type BH3 sequences and randomly substituted all other positions to generate a large number of BH3-like sequences. We modeled 20000 complex structures with Mcl-1 or Bcl-XL using the BH3-like sequences derived from five wild-type pro-apoptotic BH3 peptides. Peptide-protein interaction energies calculated from these models for each set of BH3-like sequences resulted in negatively skewed extreme value distributions. The selected BH3-like sequences from the extreme negative tail regions have highly favorable interaction energies with Mcl-1 or Bcl-XL. They are enriched in acidic and basic residues when they bind to Mcl-1 and Bcl-XL, respectively. With the charged residues often away from the binding interface, the overall electric field generated by the charged residues results in strong long-range electrostatic interaction energies between the peptide and the protein giving rise to high specificity. Cell viability studies of representative BH3-like peptides further validated the predicted specificity. This study has revealed the importance of non-hot spot residues in BH3-mimetic peptides in providing specificity to a particular anti-apoptotic protein.

Targeting AGTR1/NF-κB/CXCR4 axis by miR-155 attenuates oncogenesis in glioblastoma.

Glioblastoma (GBM) represents the most aggressive malignancy of the central nervous system. Increased expression of Angiotensin II Receptor Type 1 (AGTR1) has been associated with proliferative and infiltrative properties of glioma cells. However, the underlying mechanism of AGTR1 upregulation in GBM is still unexplored. To understand the post-transcriptional regulation of AGTR1 in GBM, we screened 3'untranslated region (3'UTR) of AGTR1 for putative miRNA binding by using prediction algorithms. Interestingly, miR-155 showed conserved binding on the 3'UTR of AGTR1, subsequently confirmed by luciferase reporter assay. Furthermore, miR-155 overexpressing GBM cells show decrease in AGTR1 expression accompanied with reduced cell proliferation, invasion, foci formation and anchorage-independent growth. Strikingly, immunodeficient mice implanted with stable miR-155 overexpressing SNB19 cells show negligible tumor growth. Notably, miR-155 attenuates NF-κB signaling downstream of AGTR1 leading to reduced CXCR4 as well as AGTR1 levels. Mechanistically, miR-155 mitigates AGTR1-mediated angiogenesis, epithelial-to-mesenchymal transition, stemness, and MAPK signaling. Similar effects were observed by using pharmacological inhibitor of IκB Kinase (IKK) complex in multiple cell-based assays. Taken together, we established that miRNA-155 post-transcriptionally regulates AGTR1 expression, abrogates AGTR1/NF-κB/CXCR4 signaling axis and elicits pleiotropic anticancer effects in GBM. This study opens new avenues for using IKK inhibitors and miRNA-155 replacement therapies for the treatment of AGTR1-positive malignancies.

Dynamics of Cellular Plasticity in Prostate Cancer Progression.

Despite the current advances in the treatment for prostate cancer, the patients often develop resistance to the conventional therapeutic interventions. Therapy-induced drug resistance and tumor progression have been associated with cellular plasticity acquired due to reprogramming at the molecular and phenotypic levels. The plasticity of the tumor cells is mainly governed by two factors: cell-intrinsic and cell-extrinsic. The cell-intrinsic factors involve alteration in the genetic or epigenetic regulators, while cell-extrinsic factors include microenvironmental cues and drug-induced selective pressure. Epithelial-mesenchymal transition (EMT) and stemness are two important hallmarks that dictate cellular plasticity in multiple cancer types including prostate. Emerging evidence has also pinpointed the role of tumor cell plasticity in driving anti-androgen induced neuroendocrine prostate cancer (NEPC), a lethal and therapy-resistant subtype. In this review, we discuss the role of cellular plasticity manifested due to genetic, epigenetic alterations and cues from the tumor microenvironment, and their role in driving therapy resistant prostate cancer.

Toxicity of exhaust particulates and gaseous emissions from gasohol (ethanol blended gasoline)-fuelled spark ignition engines.

In the last couple of decades, blending of oxygenated additives with gasoline has been advocated to reduce dependence on fossil fuels and to reduce hazardous health effects of gaseous emissions and particulate matter (PM) emitted by internal combustion (IC) engines in the transport sector worldwide. The primary objective of this research was to carry out a comparative analysis of exhaust PM emitted by gasohol (gasoline blended with 10% ethanol, v/v)-fulled spark ignition (SI) engine with that of baseline gasoline-fuelled SI engine. To assess the PM toxicity, physical, chemical and biological characterizations of PM were carried out using the state-of-the-art instruments and techniques. Measurements of regulated and unregulated gaseous species were also carried out at part/full loads. The results showed that the gasohol-fuelled engine emitted relatively lower concentrations of unregulated gaseous species such as sulfur dioxide (SO2), isocyanic acid (HNCO), etc. Physical characterization of exhaust particles revealed that the gasohol-fuelled engine emitted a significantly lower number of particles compared to the gasoline-fuelled engine. The presence of harmful polycyclic aromatic hydrocarbons (PAHs) and higher trace metal concentrations in PM emitted from the gasoline-fuelled engine was another important finding of this study. Biological characterizations showed that PM emitted from the gasohol-fuelled engine were less cytotoxic and had lower reactive oxygen species (ROS) generation potential. Mutagenicity of PM emitted from the gasohol-fuelled engine was also lower compared to that from the gasoline-fuelled engine. Overall, this study demonstrated that utilization of gasohol in SI engines led to the reduction in emissions, and lowering of PM toxicity, in addition to partial replacement of fossil fuels with renewable fuels.

A Drosophila model of oral peptide therapeutics for adult intestinal stem cell tumors.

Peptide therapeutics, unlike small-molecule drugs, display crucial advantages of target specificity and the ability to block large interacting interfaces, such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yorkie (Yki), has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian vestigial-like (VGLL) proteins, specifically their TONDU domain, competitively inhibit YAP-TEAD interaction, resulting in arrest of tumor growth. Here, we show that overexpression of the TONDU peptide or its oral uptake leads to suppression of Yki-driven intestinal stem cell tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with chromatin immunoprecipitation analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, our findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors.This article has an associated First Person interview with the first author of the paper.

Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer.

Emergence of an aggressive androgen receptor (AR)-independent neuroendocrine prostate cancer (NEPC) after androgen-deprivation therapy (ADT) is well-known. Nevertheless, the majority of advanced-stage prostate cancer patients, including those with SPINK1-positive subtype, are treated with AR-antagonists. Here, we show AR and its corepressor, REST, function as transcriptional-repressors of SPINK1, and AR-antagonists alleviate this repression leading to SPINK1 upregulation. Increased SOX2 expression during NE-transdifferentiation transactivates SPINK1, a critical-player for maintenance of NE-phenotype. SPINK1 elicits epithelial-mesenchymal-transition, stemness and cellular-plasticity. Conversely, pharmacological Casein Kinase-1 inhibition stabilizes REST, which in cooperation with AR causes SPINK1 transcriptional-repression and impedes SPINK1-mediated oncogenesis. Elevated levels of SPINK1 and NEPC markers are observed in the tumors of AR-antagonists treated mice, and in a subset of NEPC patients, implicating a plausible role of SPINK1 in treatment-related NEPC. Collectively, our findings provide an explanation for the paradoxical clinical-outcomes after ADT, possibly due to SPINK1 upregulation, and offers a strategy for adjuvant therapies.