Tumors strongly depend on their surrounding tumor microenvironment (TME) for growth and progression, since stromal elements are required to generate the optimal conditions for cancer cell proliferation, invasion, and possibly metastasis. Prostate cancer (PCa), though easily curable during primary stages, represents a clinical challenge in advanced stages because of the acquisition of resistance to anti-cancer treatments, especially androgen-deprivation therapies (ADT), which possibly lead to uncurable metastases such as those affecting the bone. An increasing number of studies is giving evidence that prostate TME components, especially cancer-associated fibroblasts (CAFs), which are the most abundant cell type, play a causal role in PCa since the very early disease stages, influencing therapy resistance and metastatic progression. This is highlighted by the prognostic value of the analysis of stromal markers, which may predict disease recurrence and metastasis. However, further investigations on the molecular mechanisms of tumor–stroma interactions are still needed to develop novel therapeutic approaches targeting stromal components. In this review, we report the current knowledge of the characteristics and functions of the stroma in prostate tumorigenesis, including relevant discussion of normal prostate homeostasis, chronic inflammatory conditions, pre-neoplastic lesions, and primary and metastatic tumors. Specifically, we focus on the role of CAFs, to point out their prognostic and therapeutic potential in PCa.

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DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here, we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, the depletion of both DHX30 isoforms leads to higher global translation but slower proliferation and lower mitochondrial energy metabolism. Isoform-specific silencing supports a role for cytoplasmic DHX30 in modulating global translation. The impact on translation and proliferation was confirmed in U2OS and MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified fourteen mitoribosome transcripts we propose as direct DHX30 targets that can be used to explore the prognostic value of this mechanism in cancer. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.

DHX30 was recently identified as a critical element of an RBP complex participating in translational control of mRNAs containing a target 3’UTR cis element, in the context of p53-dependent apoptosis. Here we show that DHX30 exhibits a more general, housekeeping function that comprises a combination of activities exerted by an isoform expressed in the cytoplasm and one, more abundant, localized in the mitochondria. Stable HCT116 cells depleted of both DHX30 isoforms showed constitutive changes in the repertoire of mRNAs associated with polysomes and enhanced translation of mRNAs coding for cytoplasmic ribosomal proteins. On the contrary, nuclear-encoded mitoribosome transcripts showed reduced translation efficiency in DHX30-depleted cells. Furthermore, HCT116 shDHX30 cells exhibited increased rRNA synthesis, higher amounts of ribosomes, and increased global translation. Surprisingly, they also showed reduced proliferation, which was also observed upon transient silencing of just the DHX30 cytoplasmic transcript. HCT116 DHX30-depleted cells showed reduced mitochondrial energy metabolism, based on oxygen consumption rate, but did not show evidence of compensatory glycolysis. Impaired mitochondrial function was also related to decreased expression of mitochondrially encoded OXPHOS components. Results were extended to U2OS and MCF7 cell lines. A gene signature comprising DHX30 and fourteen mitoribosome transcripts that are candidate direct DHX30 targets showed prognostic value in distinct cancer types from TGCA data, with higher expression being associated to reduced overall survival. Hence, it appears that DHX30 contributes to cell homeostasis through the coordination between ribosome biogenesis, global translation, and mitochondrial functions. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.

Introduction and Objective Several studies have shown that cancer-associated fibroblasts (CAFs), the most abundant component in prostate cancer (PCa) microenvironment, promote resistance to androgen deprivation therapies, and metastatic development. The aim of this study is to elucidate the mechanisms of CAF-induced therapy resistance through the establishment of in vitro 3D co-cultures. Methods In this study, the androgen-dependent PNPCa patient-derived xenograft (PDX) model (derived from soft tissue metastasis), and the androgen-independent LAPC9 PDX model (bone metastasis) are used. Epithelial and stromal cells (human and mouse origin, respectively) are separated through magnetic-associated cell sorting to generate PDX-derived organoids and fibroblasts. Fibroblast RNA and protein expression is assessed through RT-qPCR, Western Blot, and Immunofluorescence. Direct 3D co-cultures are obtained by combining fluorescently labelled organoids and fibroblasts in ultra-low attachment plates in defined media. In indirect co-cultures, organoids are cultured in Spherical Plates (Kugelmeiers) with fibroblasts on transwell inserts. Organoid viability is measured after 9 days by CellTiter-Glo 3D Assay. Results PDX-derived fibroblasts express typical CAF genes (e.g. α-smooth muscle actin and Tenascin C), and androgen receptor (AR), both at RNA and protein level. AR protein expression (as well as RNA expression of the AR target gene Fkbp5) is increased in CAFs upon treatment with dyhydrotestosterone (DHT) 10nM, while co-treatment with the AR inhibitor Enzalutamide 10mM abrogates this effect, suggestive of functional AR signaling. These CAFs can organize into tumor/CAF organoid 3D structures upon combination with PDX-derived tumor epithelial cells, providing an in vitro functional model to study tumor-stromal interactions. In indirect (transwell) co-cultures, CAFs increase the viability of the androgen dependent PNPCa organoids, but not that of androgen independent LAPC9. This growth-promoting effect is observed even upon culturing of PNPCa organoids in non-optimal growth conditions, at low DHT concentrations (DHT 0.5nM and 0.25nM versus the standard DHT 1nM). The factors mediating this process will be identified by gene expression profiling of CAFs and organoids in transwell co-cultures and/or monoculture. Conclusions Our data indicate that PCa metastatic cells modify the properties of neighbor stromal cells to support tumorigenesis, since PDX-derived fibroblasts, originally part of the mouse dermal stroma, display CAF features and active AR signaling. 3D co-cultures represent a useful tool to study the tumor-stromal interactions. Our transwell co-culture experiments suggest that CAFs play an important role in supporting the viability and growth of early metastatic androgen dependent PCa cells (PNPCa) by secreting pro-tumorigenic factors. The possibility that CAFs exert different effects depending on the tumor stage setting (early vs advanced) will be subsequently investigated. Citation Format: Francesco Bonollo, Natalie Sampson, George Thalmann, Sofia Karkampouna, Marianna Kruithof-de Julio. Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in 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 B008.