Second generation PSMA-targeted turn-on probe for imaging cargo release in prostate cancer cells.

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism occurs. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, we examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, we provide novel evidence that efficient endocytosis of EGF-EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, this is the first report demonstrating that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF-EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines.

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.

Otto Warburg was the first to postulate a role for cell metabolism in carcinogenesis. Hanahan and Weinberg recently updated their seminal review to include metabolic reprogramming as a hallmark of cancer.1 While normal cells predominantly depend on mitochondrial oxidative phosphorylation for their energy needs, cancer cells favor aerobic glycolysis, also known as the Warburg effect. This unique metabolic shift provides a survival advantage to the cancer cells in the developing tumor microenvironment and, paradoxically, provides oncologists with potential therapeutic targets. Indeed, metabolic changes have been described as the “Achilles’ heel” of cancer.2 One such metabolic change is the acidification of the tumor microenvironment by carbonic anhydrases (CAs), especially CAIX. CAIX expression is regulated by the pro-survival transcription factor hypoxia-inducible factor-1α (HIF-1α). CAIX is overexpressed in many tumor types and has been linked to poor prognosis, purportedly due to its involvement in the breakdown of extracellular matrix, protease, and growth factor activation and augmentation of metastatic potential. Previous research has focused predominantly on the metabolic and molecular features of tumor cells, but there is an increasing awareness that stromal cells recruited to the tumor microenvironment are important contributors to the development, progression, and aggressiveness of tumors. In the June 1, 2013 issue of Cell Cycle, Chiarugi and colleagues demonstrated the role of CAIX-expressing cancer-associated fibroblasts (CAFs) in regulating the epithelial–mesenchymal transition (EMT) of prostate cancer cells.3 They report that normal human prostate fibroblasts (HPFs) do not express CAIX; however, exposing HPF to conditioned media (CM) from prostate cancer (PCa) cells activates HPF cells to CAFs. CAIX expression was also induced in prostate cancer (PCa) cells treated with CM from CAFs, highlighting the cross-talk between the tumor and its microenvironment (Fig. 1). Interestingly, CAIX was expressed at similar levels in CAFs and serum-starved PCa cells, but PCa cells treated with CM from CAFs expressed higher CAIX levels than CAFs themselves. However, CAIX activity was higher in CAFs compared with PCa cells treated with CAF CM. CAIX expression in both PCa cells and CAFs was HIF-1α-dependent despite these experiments being conducted under normoxic conditions; this observation further supports that the activation of HIF1α signaling was mediated by redox-based stabilization of HIF1α.4 CAIX inhibition decreased extracellular acidification thereby demonstrating that CAIX is necessary and sufficient for such acidification. Figure 1. Tumor microenvironmental cross-talk mediates epithelial–mesenchymal transition. (1) Malignant transformation induces the secretion of growth factors; these growth factors activate resident and recruited fibroblasts to CAFs and ... The role of matrix metalloproteinases (MMPs) in aggressive/metastatic disease and their response to low pH are well documented.5 Consequently, the authors investigated the link between CAIX and MMP expression. CAIX-induced acidosis increased the expression of MMPs in CAFs, and inhibition of CAIX decreased the secretion of MMP-2 and MMP-9. Inhibition of MMPs reduced the invasiveness of PCa cells. Addition of recombinant MMPs to CAIX inhibited CM rescues ability of PCA cells to undergo EMT. In immune-compromised mice, inhibition of CAIX in CAFs reduced the ability of PCa cells to form viable tumors and effectively metastasise to the lung. The cellular and mechanistic insights provided by this article are exciting and timely, but it is important that these insights be applied in patient samples to understand the clinical significance of the findings. We have previously reported, in 2 independent head and neck cancer cohorts, that stromal CAIX levels are more strongly associated with poor survival than tumor CAIX.6,7 High-stromal CAIX was also associated with increased nodal metastasis.7 However, we did not identify the specific contributing stromal cell-types. In the future, co-staining tissue micro-arrays with α-smooth muscle actin (a specific marker for CAFs) would potentially improve the definition of the stromal contribution to CAIX expression and association with prognosis. Chiarugi and colleagues report CAFs as the main protagonists in the CAIX-induced tumor aggressiveness, but the role of other cell types in the tumor microenvironment should be investigated. Furthermore, the direct effect of CAIX inhibition in PCa cells needs to be determined. CAIX is an attractive therapeutic target, because its expression is relatively tumor specific, several low-toxicity pharmaceuticals are available, and novel analogs of existing inhibitors are currently being tested.8 Given the disappointing results of MMP inhibition trials,5 targeted reduction of MMP2 and MMP9 by inhibition of CAIX may provide an alternative strategy. Also, the effects of CAIX inhibition on other MMPs and the potential for regulation by other compensatory mechanisms should be addressed.

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.

RANKL-elicited RANK activity plays critical roles in many biological and pathological conditions, including osteoclast differentiation/bone remodeling, lymph node/thymic development, central thermoregulation and progesterone-driven mammary gland maturation, differentiation and carcinogenesis. RANKL can be derived from osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. We previously showed that malignant prostate cancer (PCa) cells expressed RANKL and that its expression was correlated with clinical PCa progression and bone metastasis. RANKL expression at the single cell level in primary PCa specimens predicts PCa patient survival. We demonstrated RANKL overexpression promoted epithelial-to-mesenchymal transition (EMT), stem cells, and neuroendocrine (NE) phenotypes of PCa cells through transactivation of c-Myc/Max and AP4 via RANK-mediated signaling and conferred PCa bone and soft tissue metastases in mice. Blockade of RANKL-RANK signaling as well as their downstream regulators abolished the metastasis of PCa induced by RANKL. Furthermore, recombinant RANKL protein alone is sufficient to induce bone colonization and growth of RANKL- and non-metastatic PCa and breast cancer cells. Even a few RANKL+ PCa cells are sufficient to initiate the in vivo metastatic cascade by recruiting non-tumorigenic RANKL- PCa cells to participate in the metastatic process via downstream signaling amplification, implicating metastasis-initiating properties of RANKL-expressing PCa cells. Recently, we further demonstrated that RANKL rendered metastatic potential to breast, lung, renal, and liver cancer cells and promote their metastases to bone and other soft tissues in mice. Collectively, these results demonstrated RANKL is a uniform factor that drives bone metastasis of many different cancers, including prostate, breast, lung, renal, and liver cancers. The autocrine/paracrine RANKL-RANK signaling in cancer cells establishes a premetastatic niche through a “vicious cycle”, inducing RANKL and c-Met expression via activation of c-Myc/Max, and this promotes PCa EMT progression, stem and NE cell properties, and bone and soft tissue metastases. RANKL-expressing cancer cells possess metastasis-initiating potential that recruits and reprograms bystander non-tumorigenic cells to participate in the metastatic process. RANKL expression status therefore offers new insights for dissecting the mechanism by which PCa cells exhibit propensity for bone colonization/metastasis. Citation Format: Gina Chia-Yi Chu, Haiyen E. Zhau, Ruoxiang Wang, Leland W.K. Chung. Autocrine/paracrine RANKL-RANK signaling promotes cancer bone metastasis and establishes premetastatic niche recruiting bystander cancer cells to participate in the metastatic process. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B42.

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Simple SummaryMetastatic castration-resistant PCa (mCRPC) is a clinically highly lethal disease; the mechanisms underlying the lethal disease remain poorly understood. Furthermore, no effective treatment for cancer metastasis exists. In this study, we have demonstrated that prostate cancer cells required bone marrow-derived cells for their growth, survival and metastasis to the host bone marrow. Our findings have provided new evidence suggesting that cancer cell-specific signals may mediate interactions between prostate cancer cells and bone marrow cells during progression of mCRPC. Therapeutic interventions using a selective inhibitor of lipid kinase PIP5K1α may not only inhibit the growth of primary tumors but may also target the lethal mCRPC within tumor-microenvironment.Cancer cells facilitate growth and metastasis by using multiple signals from the cancer-associated microenvironment. However, it remains poorly understood whether prostate cancer (PCa) cells may recruit and utilize bone marrow cells for their growth and survival. Furthermore, the regulatory mechanisms underlying interactions between PCa cells and bone marrow cells are obscure. In this study, we isolated bone marrow cells that mainly constituted populations that were positive for CD11b and Gr1 antigens from xenograft PC-3 tumor tissues from athymic nu/nu mice. We found that the tumor-infiltrated cells alone were unable to form tumor spheroids, even with increased amounts and time. By contrast, the tumor-infiltrated cells together with PCa cells formed large numbers of tumor spheroids compared with PCa cells alone. We further utilized xenograft athymic nu/nu mice bearing bone metastatic lesions. We demonstrated that PCa cells were unable to survive and give rise to colony-forming units (CFUs) in media that were used for hematopoietic cell colony-formation unit (CFU) assays. By contrast, PC-3M cells survived when bone marrow cells were present and gave rise to CFUs. Our results showed that PCa cells required bone marrow cells to support their growth and survival and establish bone metastasis in the host environment. We showed that PCa cells that were treated with either siRNA for PIP5K1α or its specific inhibitor, ISA-2011B, were unable to survive and produce tumor spheroids, together with bone marrow cells. Given that the elevated expression of PIP5K1α was specific for PCa cells and was associated with the induced expression of VEGF receptor 2 in PCa cells, our findings suggest that cancer cells may utilize PIP5K1α-mediated receptor signaling to recruit growth factors and ligands from the bone marrow-derived cells. Taken together, our study suggests a new mechanism that enables PCa cells to gain proliferative and invasive advantages within their associated host microenvironment. Therapeutic interventions using PIP5K1α inhibitors may not only inhibit tumor invasion and metastasis but also enhance the host immune system.

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 microm) or the Akt-specific inhibitor-X (Akti-X; 2.5 microm). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.

Simple SummaryProstate cancer (PCa) metastasizes preferentially to the bone marrow where it becomes difficult to treat. PCa cells in the bone marrow may survive, dormant and undetected for many years before patients eventually relapse with metastatic disease. Bone marrow is a complex tissue that initially is hostile to the PCa cells, Understanding how cancer cells survive in the bone marrow and what changes to the bone microenvironment permit them to switch to an actively growing state could offer new therapeutic strategies to combat metastatic PCa. In this study, we describe a method to culture PCa cells with two other cell types from the bone marrow, stromal cells and endothelial cells, as a way to study the interactions among these cell types. We found that factors produced by bone marrow endothelial cells, but not endothelial cells from other tissues, trigger PCa cells to either die or enter a dormant state, similar to what has been observed in patients when PCa cells initially colonize the bone marrow. Further analysis of the cell interactions within the culture model described in this study will offer increased understanding of PCa interaction with the bone marrow environment.The bone marrow tumor microenvironment (BMTE) is a complex network of cells, extracellular matrix, and sequestered signaling factors that initially act as a hostile environment for disseminating tumor cells (DTCs) from the cancerous prostate. Three-dimensional (3D) culture systems offer an opportunity to better model these complex interactions in reactive stroma, providing contextual behaviors for cancer cells, stromal cells, and endothelial cells. Using a new system designed for the triculture of osteoblastic prostate cancer (PCa) cells, stromal cells, and microvascular endothelial cells, we uncovered a context-specific pro-apoptotic effect of endothelial cells of the bone marrow different from those derived from the lung or dermis. The paracrine nature of this effect was demonstrated by observations that conditioned medium from bone marrow endothelial cells, but not from dermal or lung endothelial cells, led to PCa cell death in microtumors grown in 3D BMTE-simulating hydrogels. Analysis of the phosphoproteome by reverse phase protein analysis (RPPA) of PCa cells treated with conditioned media from different endothelial cells identified the differential regulation of pathways involved in proliferation, cell cycle regulation, and apoptosis. The findings from the RPPA were validated by western blotting for representative signaling factors identified, including forkhead box M1 (FOXM1; proliferation factor), pRb (cell cycle regulator), and Smac/DIABLO (pro-apoptosis) among treatment conditions. The 3D model presented here thus presents an accurate model to study the influence of the reactive BMTE, including stromal and endothelial cells, on the adaptive behaviors of cancer cells modeling DTCs at sites of bone metastasis. These findings in 3D culture systems can lead to a better understanding of the real-time interactions among cells present in reactive stroma than is possible using animal models.

Gefitinib (Iressa)–a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase–has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells.

Elevated oxidative stress is more frequently observed in cancer cells than in normal cells. Thus, further exposure to exogenous reactive oxygen species (ROS) is expected to push cancer to cell death, whereas normal cells may maintain redox homeostasis through ROS mediated adaptive responses. We previously demonstrated that parthenolide selectively enhances radiosensitivity of prostate cancer cells through activation of a NADPH oxidase-dependent redox signaling pathway. Our present study identifies the status of the redox sensitive Kelch-like ECH-associated protein-1 (Keap1) as a central regulator responsible for the parthenolide-mediated differential redox modification. This results in both a radiosensitization effect in prostate cancer cells and a radioresistance effect in normal prostate epithelial cells. Mechanistically, parthenolide increases the reduced state of Keap1 in prostate cancer cells in a thioredoxin-dependent manner but causes oxidation of Keap1 in normal prostate epithelial cells. Selective knock-down of thioredoxin leads to Keap1 oxidation and subsequent activation of the NF-E2-related factor 2 (Nrf2). These results suggest that redox modification of Keap1 is a major mechanism for the parthenolide effect on radiation therapy in cancer and normal cells. Consistent with the differential effect of parthenolide in cancer and normal cells, the levels of superoxide, hydrogen peroxide and peroxynitrite were selectively increased in parthenolide treated prostate cancer cells but not in normal prostate epithelial cells, indicating the selective increase of ROS in cancer cells. Parthenolide reduces the levels of Nrf2 targets, antioxidant proteins including manganese superoxide dismutase and heme oxygenase in prostate cancer cells, but increases the levels of these proteins in normal cells. This suggests that an antioxidant response is critical for the protection of normal cells against ROS generated from parthenolide. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2073. doi:1538-7445.AM2012-2073

Background: Androgens stimulate androgen receptor (AR) signaling, driving prostate cancer (PC) cell proliferation and progression. Androgen deprivation therapy (ADT) effectively treats primary disease, but 20-30% of patients relapse after ~5 years, resulting in castration-resistant prostate cancer (CRPC), which has limited treatment options. We have previously shown in a murine model of CRPC that macrophages promote AR signaling in CRPC and that depleting tumor macrophages extends survival in response to ADT with Lupron. This correlates with reduced androgen levels in macrophage-depleted tumors and the ability of macrophages to transfer cholesterol, the anabolic precursor of androgens, to prostate cancer cells. However, the mechanism by which this transfer occurs, and whether cholesterol exchange mediates resistance to ADT, is currently unknown. Methods: To assess cholesterol transfer from macrophages to cancer cells, we established a culture system involving prostate cancer cell lines and either the RAW264.7 macrophage cell line or bone marrow-derived macrophages (BMDMs). Macrophages were pre-loaded with cholesterol using fluorescently labeled low-density lipoprotein (LDL) and were co-cultured or placed in a transwell system with tumor cells for 24 hours. Cholesterol transfer was then measured by flow cytometry. Results: Fluorescent signal from LDL was detected in 50-70% of tumor cells after 24 hours of co-culture. Cell contact was required for cholesterol transfer, as no fluorescence was detected in prostate cancer cells during transwell experiments. Macrophages were also able to internalize and transfer acetylated LDL, which is poorly taken up by tumor cells compared to unmodified LDL. Macrophage polarization with interleukin-4 did not impact cholesterol transfer and uptake of acetylated LDL occurred independently of the scavenger receptor CD36. The scavenger receptor SR-B1, which is often upregulated in prostate cancer cells, was dispensable for transfer, as knocking out SR-B1 via CRISPR/Cas9 in cancer cell lines did not reduce LDL uptake. Conclusion: Cholesterol transfer between macrophages and prostate cancer cells occurs in a contact-dependent manner, independently of the scavenger receptors most commonly associated with cholesterol accumulation. Citation Format: Olabisi Osunmakinde, Brian Ruffell. Investigating cholesterol transfer between macrophages and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2881.

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Screening and biomarker identification have improved prostate cancer outcomes, but prostate cancer progression remains a critical challenge.Mitochondria have garnered increasing attention in prostate and other cancers, and metabolic and non-metabolic mitochondrial contributions to cancer merit further investigation.In their study, Furnish and colleagues found that mitochondrial Rho GTPase 2 (MIRO2) expression is upregulated in recurrent and progressive prostate cancer, and that MIRO2 depletion via shRNA abrogates prostate cancer cell growth in vitro and in vivo.The authors identified MIRO2 protein interactors using MIRO2 immunoprecipitation and mass spectrometry and revealed general control nonderepressible (GCN1) is a MIRO2 binding partner.Mechanistically, the authors showed that MIRO2 facilitates GCN1-mediated activation of GCN2, which results in eukaryotic translation initiation factor 2 alpha (eIF2a) phosphorylation and transcriptional upregulation of transcriptional activator 4 (ATF4).Immunohistochemistry data from prostate cancer xenografts implicated HIF1a as an oncogenic MIRO2 regulator.Overall, this work presents a previously unknown mechanism by which mitochondria promote prostate cancer progression and suggests that MIRO2 could be a useful therapeutic target for recurrent and progressive prostate cancer.