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.

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.

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.

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.

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.

Proproliferative function of adaptor protein GRB10 in prostate carcinoma.

Growth factor receptor-binding protein 10 (GRB10) is a well-known adaptor protein and a recently identified substrate of the mammalian target of rapamycin (mTOR). Depletion of GRB10 increases insulin sensitivity and overexpression suppresses PI3K/Akt signaling. Because the major reason for the limited efficacy of PI3K/Akt-targeted therapies in prostate cancer (PCa) is loss of mTOR-regulated feedback suppression, it is therefore important to assess the functional importance and regulation of GRB10 under these conditions. On the basis of these background observations, we explored the status and functional impact of GRB10 in PCa and found maximum expression in phosphatase and tensin homolog (PTEN)-deficient PCa. In human PCa samples, GRB10 inversely correlated with PTEN and positively correlated with pAKT levels. Knockdown of GRB10 in nontumorigenic PTEN null mouse embryonic fibroblasts and tumorigenic PCa cell lines reduced Akt phosphorylation and selectively activated a panel of receptor tyrosine kinases. Similarly, overexpression of GRB10 in PTEN wild-type PCa cell lines accelerated tumorigenesis and induced Akt phosphorylation. In PTEN wild-type PCa, GRB10 overexpression promoted mediated PTEN interaction and degradation. PI3K (but not mTOR) inhibitors reduced GRB10 expression, suggesting primarily PI3K-driven regulation of GRB10. In summary, our results suggest that GRB10 acts as a major downstream effector of PI3K and has tumor-promoting effects in prostate cancer.-Khan, M. I., Al Johani, A., Hamid, A., Ateeq, B., Manzar, N., Adhami, V. M., Lall, R. K., Rath, S., Sechi, M., Siddiqui, I. A., Choudhry, H., Zamzami, M. A., Havighurst, T. C., Huang, W., Ntambi, J. M., Mukhtar, H. Proproliferatve function of adaptor protein GRB10 in prostate carcinoma.

Entropically driven controlled release of paclitaxel from poly(2-ethyl-2-oxazoline) coated maghemite nanostructures for magnetically guided cancer therapy.

Theranostic nanostructures serve a dual purpose of therapy and diagnosis. A major fraction of these are based on polymer coated magnetic nanostructures of iron oxides (magnetite and maghemite), owing to the efficient drug loading capacity of polymer shells and enhanced magnetic contrast effects of the iron oxide core. In the current work we are proposing poly(2-ethyl-2-oxazoline) coated linear thermoresponsive nanostructures of maghemite (γ-Fe2O3) for potential application in targeted cancer therapy. The polymer coating was obtained via a modified sol-gel technique based on entropically driven phase separation of poly(2-ethyl-2-oxazoline) above its cloud point (CP) temperature of 63 °C in water. The developed nanostructures were further loaded with paclitaxel, a polar anticancer compound at room temperature (25 °C). The entropically driven release of paclitaxel at various concentrations and physiological temperatures was modeled and their application to the PC3 prostrate cancer cell line was investigated by treating in vitro. The steering efficiency of the magnetic nanostructures during their navigation through large blood vessels was also analyzed with the help of a synthetic model of the human axillary artery. The proposed application of these newly developed nanostructures can easily be extended towards localized delivery of additional polar anticancer drugs like cisplatin and doxorubicin.