Advanced prostate cancer initially responds to hormonal treatment, but ultimately becomes resistant and requires more potent therapies. One mechanism of resistance observed in around 10–20% of these patients is lineage plasticity, which manifests in a partial or complete small cell or neuroendocrine prostate cancer (NEPC) phenotype. Here, we investigate the role of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex in NEPC. Using large patient datasets, patient-derived organoids and cancer cell lines, we identify mSWI/SNF subunits that are deregulated in NEPC and demonstrate that SMARCA4 (BRG1) overexpression is associated with aggressive disease. We also show that SWI/SNF complexes interact with different lineage-specific factors in NEPC compared to prostate adenocarcinoma. These data point to a role for mSWI/SNF complexes in therapy-related lineage plasticity, which may also be relevant for other solid tumors.
The actin cytoskeleton is an attractive target for bacterial toxins. The ADP-ribosyltransferase TccC3 from the insect bacterial pathogen Photorhabdus luminescence modifies actin to force its aggregation. We intended to transport the catalytic part of this toxin preferentially into cancer cells using a toxin transporter (Protective antigen, PA) which was redirected to Epidermal Growth Factor Receptors (EGFR) or to human EGF receptors 2 (HER2), which are overexpressed in several cancer cells. Protective antigen of anthrax toxin forms a pore through which the two catalytic parts (lethal factor and edema factor) or other proteins can be transported into mammalian cells. Here, we used PA as a double mutant (N682A, D683A; mPA) which cannot bind to the two natural anthrax receptors. Each mutated monomer is fused either to EGF or to an affibody directed against the human EGF receptor 2 (HER2). We established a cellular model system composed of two cell lines representing HER2 overexpressing esophageal adenocarcinomas (EACs) and EGFR overexpressing esophageal squamous cell carcinomas (ESCCs). We studied the specificity and efficiency of the re-directed anthrax pore for transport of TccC3 toxin and established Photorhabdus luminescence TccC3 as a toxin suitable for the development of a targeted toxin selectively killing cancer cells.
Advanced prostate cancer initially responds to hormonal treatment, but ultimately becomes resistant and requires more potent therapies. One mechanism of resistance observed in ~10% of these patients is through lineage plasticity, which manifests in a partial or complete small cell or neuroendocrine prostate cancer (NEPC) phenotype. Here, we investigate the role of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex in NEPC. Using large patient datasets, patient-derived organoids and cancer cell lines, we identify mSWI/SNF subunits that are deregulated in NEPC and demonstrate that SMARCA4 (BRG1) overexpression is associated with aggressive disease. We also show that SWI/SNF complexes interact with different lineage-specific factors in NEPC compared to prostate adenocarcinoma. These data suggest a role for mSWI/SNF complexes in therapy-related lineage plasticity, which may be relevant for other solid tumors.
Prostate cancer is the most common malignancy, accounting for about 25% of all incident cases among men in industrialized countries. The human androgen-dependent prostate cancer cell line LNCaP, which is derived from a metastatic lesion of human prostatic adenocarcinoma, is frequently used to study prostate cancer associated signaling pathways in vitro. Recently it was described that Rho GTPase activation in these cells leads to apoptotic responses. We used the bacterial toxins CNFy and CNF1, which specifically and directly activate Rho GTPases by deamidation of a single glutamine. We asked whether these Rho activators could induce apoptosis in LNCaP cells. Our results indicate that RhoA activation, induced by CNFy, does lead to intrinsic apoptosis of the cells. Analysis of the underlying signaling pathway reveals that apoptosis induction requires the activity of Rho kinase (ROCK) and myosin activation, an apoptotic pathway previously identified in cancer stem cells.
Here we explored the role of minor spliceosome (MiS) function and minor intron-containing gene (MIG) expression in prostate cancer (PCa). We show MIGs are enriched as direct interactors of cancer-causing genes and their expression discriminates PCa progression. Increased expression of MiS U6atac snRNA, including others, and 6x more efficient minor intron splicing was observed in castration-resistant PCa (CRPC) versus primary PCa. Notably, androgen receptor signalling influenced MiS activity. Inhibition of MiS through siU6atac in PCa caused minor intron mis-splicing and aberrant expression of MIG transcripts and encoded proteins, which enriched for MAPK activity, DNA repair and cell cycle. Single cell-RNAseq confirmed cell cycle defects and lineage dependency on the MiS from primary to CRPC and neuroendocrine PCa. siU6atac was ~50% more efficient in lowering tumor burden of CRPC cells and organoids versus current state-of-the-art combination therapy. In all, MiS is a strong therapeutic target for lethal PCa and potentially other cancers.
The role of major intron splicing in PCa is well established and highly investigated. However, the equally important and impactful minor spliceosome (MiS) and minor intron splicing have remained underexplored in PCa. Here for the first time, we show that minor intron containing genes (MIGs) are enriched as direct interactors of PCa-causing genes and that their expression levels correspond to different stages of PCa progression. Accordingly, we show that U6atac expression and MiS activity reinforces cell growth and correlates with PCa progression. Consequently, highly proliferative PCa cells are strongly susceptible to siU6atac-mediated MiS inhibition. In this sense, siU6atac is significantly more efficient in lowering the tumor burden of castration resistant prostate cancer (CRPC) cells and organoids than the current state-of-the-art combination therapy. Mechanistically, we show that inhibition of the MiS through siU6atac causes minor and major intron mis-splicing and aberrant expression of MIG and non-MIG transcripts and encoded proteins, which enriches for MAPK activity, DNA repair, and cell cycle. Single cell-RNAseq confirms cell cycle defects and lineage dependency on the MiS from primary to CRPC and neuroendocrine PCa. Finally, our data provides strong evidence that Androgen receptor signaling functions as a regulator of MiS activity throughout PCa disease progression to CRPC while p38MAPK influences MiS activity in neuroendocrine PCa. Furthermore, we discovered that MiS inhibition in neuroendocrine PCa shifts alternative splicing in favor of canonical REST1 isoform thereby blocking lineage plasticity and progression to lethal therapy resistant PCa. This finding positions the MiS as yet another disruptor of a crucial molecular pathway underpinning the emergence of CRPC and neuroendocrine PCa. Based on these findings we posit that the MiS may be a common denominator of prominent PCa driver genes and a driving factor of advanced lethal PCa. We hypothesize that the MiS represents a point of vulnerability in therapy-resistant PCa. Citation Format: Anke Augspach, Mark Andrew Rubin, Rahul Kanadia. Minor intron splicing is critical for survival of lethal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A013.
Prostate cancer represents one of the leading causes of morbidity and mortality of men worldwide. In precision medicine, tumors are screened for specific genetic alterations known as predictive markers for targeted therapy. In androgen-independent prostate cancer cells and in tissue samples of a prostate cancer patient treated with Goserelin, we identified the self-activating splice variant Rac1b. Importantly, the expression of Rac1b was sufficient to induce AR-dependent gene synthesis. We hypothesized that Rac1b antagonizes androgen depletion induced cancer cell death by blocking pro-apoptotic signalling pathways. In line with that selective knockdown of Rac1b or inhibition of Rac-dependent signalling pathways reinduced apoptosis in androgen-independent prostate cancer cells suggesting Rac1b inhibition as a potential novel therapeutic add on strategy against prostate cancer.
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