Upregulation of Succinate Dehydrogenase (SDHA) Contributes to Enhanced Bioenergetics of Ovarian Cancer Cells and Higher Sensitivity to Anti-Metabolic Agent Shikonin

We discovered that the overexpression of mitochondrial enzyme succinate dehydrogenase (SDHA) is particularly prevalent in ovarian carcinoma and promotes highly metabolically active phenotype. Succinate dehydrogenase deficiency has been previously studied in some rare disorders. However, the role of SDHA upregulation and its impact on ovarian cancer metabolism has never been investigated, emphasizing the need for further research. We investigated the functional consequences of SDHA overexpression in ovarian cancer. Using proteomics approaches and biological assays, we interrogated protein content of metabolic pathways, cell proliferation, anchorage-independent growth, mitochondrial respiration, glycolytic function, and ATP production rates in those cells. Lastly, we performed a drug screening to identify agents specifically targeting the SDHA overexpressing tumor cells. We showed that SDHA overexpressing cells are characterized by enhanced energy metabolism, relying on both glycolysis and oxidative phosphorylation to meet their energy needs. In addition, SDHA-high phenotype was associated with cell vulnerability to glucose and glutamine deprivation, which led to a substantial reduction of ATP yield. We also identified an anti-metabolic compound shikonin with a potent efficacy against SDHA overexpressing ovarian cancer cells. Our data underline the unappreciated role of SDHA in reprogramming of ovarian cancer metabolism, which represents a new opportunity for therapeutic intervention

Targeting a mimotope vaccine to activating FcγFc\gamma receptors empowers dendritic cells to prime specific CD8+CD8^{+} T cell responses in tumor-bearing mice

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcγ2a) to deliver the antigenic cassette to the activating Fcγ receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcγ2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcγ2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies

Patient-Derived Xenografts of High-Grade Serous Ovarian Cancer Subtype as a Powerful Tool in Pre-Clinical Research

(1) Background. PDX models have become the preferred tool in research laboratories seeking to improve development and pre-clinical testing of new drugs. PDXs have been shown to capture the cellular and molecular characteristics of human tumors better than simpler cell line-based models. More recently, however, hints that PDXs may change their characteristics over time have begun to emerge, emphasizing the need for comprehensive analysis of PDX evolution. (2) Methods. We established a panel of high-grade serous ovarian carcinoma (HGSOC) PDXs and developed and validated a 300-SNP signature that can be successfully utilized to assess genetic drift across PDX passages and detect PDX contamination with lymphoproliferative tissues. In addition, we performed a detailed histological characterization and functional assessment of multiple PDX passages. (3) Results. Our data show that the PDXs remain largely stable throughout propagation, with marginal genetic drift at the time of PDX initiation and adaptation to mouse host. Importantly, our PDX lines retained the major histological characteristics of the original patients’ tumors even after multiple passages in mice, demonstrating a strong concordance with the clinical responses of their corresponding patients. (4) Conclusions. Our data underline the value of defined HGSOC PDXs as a pre-clinical tumor model

An unbiased seed-based RNAi selection screen identifies small RNAs that inhibit androgen signaling and prostate cancer cell growth

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration-resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here, we explore a therapeutic strategy for PCa based on the ability of shRNAs/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3′ UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell-cycle genes. When tested in mice, the toxic shRNAs also inhibited castration-resistant PCa and exhibited therapeutic efficacy in pre-established tumors. Our findings highlight RNAi of androgen signaling networks as a promising therapeutic strategy for PCa

Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir