Metastatic Prostate Cancer Cells Research Articles

6667 IGF1 Induces The Expression of CYR61 In Metastatic Prostate Cancer

Abstract Disclosure: G.L. Ortiz Hernández: None. M. Walker: None. L. Woods-Burnham: None. R.A. Kittles: None. C.A. Casiano: None. S.L. Neuhausen: None. The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Our goal was to assess the contribution of the interaction of CYR61-IGF1 to PCa cell proliferation. Their combined role may explain a proportion of aggressive PCa. This study was designed to characterize the role of IGF1-induced expression of CYR61 in metastatic PCa cellular models and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, migration, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. In addition, we found that IGF1 induces protein expression of CYR61 in a time-dependent manner, validated by both immunoblotting and confocal microscopy. PC3 cell lines are androgen receptor negative (AR-) and glucocorticoid receptor positive (GR+), which is characteristic of aggressive, advanced metastatic castration-resistant prostate cancer (mCRPC). Currently, we are conducting the same experiments in LNCaP cells, which are AR+/GR-, to determine if the CYR61-IGF1 interplay also occurs in PCa cells capable of AR signaling. In conclusion, we found that CYR61 expression is induced by IGF1 and its inhibition decreased the capacity of metastatic PCa cells to proliferate. Our results suggest that CYR61 inhibition may have the potential for treating metastatic PCa. Presentation: 6/1/2024

6667 IGF1 Induces the Expression of CYR61 in Metastatic Prostate Cancer

Abstract Disclosure: G.L. Ortiz Hernández: None. M. Walker: None. L. Woods-Burnham: None. R.A. Kittles: None. C.A. Casiano: None. S.L. Neuhausen: None. The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Our goal was to assess the contribution of the interaction of CYR61-IGF1 to PCa cell proliferation. Their combined role may explain a proportion of aggressive PCa. This study was designed to characterize the role of IGF1-induced expression of CYR61 in metastatic PCa cellular models and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, migration, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. In addition, we found that IGF1 induces protein expression of CYR61 in a time-dependent manner, validated by both immunoblotting and confocal microscopy. PC3 cell lines are androgen receptor negative (AR-) and glucocorticoid receptor positive (GR+), which is characteristic of aggressive, advanced metastatic castration-resistant prostate cancer (mCRPC). Currently, we are conducting the same experiments in LNCaP cells, which are AR+/GR-, to determine if the CYR61-IGF1 interplay also occurs in PCa cells capable of AR signaling. In conclusion, we found that CYR61 expression is induced by IGF1 and its inhibition decreased the capacity of metastatic PCa cells to proliferate. Our results suggest that CYR61 inhibition may have the potential for treating metastatic PCa. Presentation: 6/1/2024

Abstract C098: CYR61 expression in prostate cancer patients at City of Hope

Abstract In the US, African-American (AA) and Hispanic/Latino (H/L) men have higher rates of advanced prostate cancer (PCa), increased PCa-specific mortality, and are more likely to undergo metastasis than non-Hispanic White (NHW) men attributed to a lack of access to early screening, and a higher prevalence of risk factors such as obesity, diet, and genetic differences. Consequently, effective treatment of advanced PCa and elimination of racial and ethnic disparities for this malignancy require the identification of mechanisms of disease progression. In PCa, the cysteine-rich angiogenic inducer 61 (CYR61) contributes to malignant cell growth through interactions with extracellular ligands, such as integrins avb3 and a6b4. In previous studies, we have found that CYR61 is upregulated in metastatic PCa cell lines and knockdown of this protein significantly reduced metastatic cell proliferation, viability, prostasphere formation, and activation of the PI3/AKT pathway, indicating a potential utilization of CYR61 inhibition in the therapy of metastatic PCa. Interestingly, we have observed that in metastatic PCa cell models, IGF1 induction increased CYR61 protein expression. However, the correlation between PCa aggressiveness and levels of CYR61 in tumor-derived tissues from patients is poorly studied. Our goal with this study is to identify possible correlations in CYR61 expression that may reflect health disparities in PCa outcomes. We used in silico analyses from whole exome paired tumor/normal DNA sequencing and tumor RNA sequencing data available on 542 PCa cases in the City of Hope POSEIDON database to determine whether gene expression of CYR61 and IGF1 was correlated with genetic ancestry, Gleason score, and tumor purity. In univariate analyses, we found that H/L PCa cases had significantly higher levels of expression of CYR61 than NHW PCa cases (p = 0.043) and that both H/L and AA PCa cases had higher IGF1 expression levels, although only significant in AA (p=0.031). We also found that PCa patients with Gleason scores of 8 and higher had significantly lower expression of IGF1 than those with Gleason scores of 7 or less (p=0.006). Both CYR61 and IGF1 expression levels were significantly lower in those with luminal B (p=0.00000000053, p=0.000009, respectively) and with basal subtypes (p=0.00035, p=0.000069, respectively compared to luminal A subtype. Tumor purity was estimated by RNA-seq based on 141 genes specific to stroma and 141 genes specific to immune cells. CYR61 expression was significantly correlated with stromal and immune infiltration. These results suggest that reduced CYR61 expression is associated with more aggressive PCa. Additional analyses are ongoing to understand the interplay of variables of race and ethnicity, age, Gleason score, and tumor purity and to investigate outcomes of biochemical recurrence and death from PCa. Citation Format: Greisha L. Ortiz-Hernandez, Yuan Chun Ding, Susan L. Neuhausen. CYR61 expression in prostate cancer patients at City of Hope [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C098.

Liver X Receptors Enhance Epithelial to Mesenchymal Transition in Metastatic Prostate Cancer Cells.

Prostate cancer (PCa) is one of the most common cancers in men. Metastasis is the leading cause of death in prostate cancer patients. One of the crucial processes involved in metastatic spread is the "epithelial-mesenchymal transition" (EMT), which allows cells to acquire the ability to invade distant organs. Liver X Receptors (LXRs) are nuclear receptors that have been demonstrated to regulate EMT in various cancers, including hepatic cancer. Our study reveals that the LXR pathway can control pro-invasive cell capacities through EMT in prostate cancer, employing ex vivo and in vivo approaches. We characterized the EMT status of the commonly used LNCaP, DU145, and PC3 prostate cancer cell lines through molecular and immunohistochemistry experiments. The impact of LXR activation on EMT function was also assessed by analyzing the migration and invasion of these cell lines in the absence or presence of an LXR agonist. Using in vivo experiments involving NSG-immunodeficient mice xenografted with PC3-GFP cells, we were able to study metastatic spread and the effect of LXRs on this process. LXR activation led to an increase in the accumulation of Vimentin and Amphiregulin in PC3. Furthermore, the migration of PC3 cells significantly increased in the presence of the LXR agonist, correlating with an upregulation of EMT. Interestingly, LXR activation significantly increased metastatic spread in an NSG mouse model. Overall, this work identifies a promoting effect of LXRs on EMT in the PC3 model of advanced prostate cancer.

The recovery from taxane mediated apoptosis in PC-3 castration-resistant metastatic prostate cancer cells

Here, we revealed the reversibility of cabazitaxel (CBZ)-induced apoptosis in PC-3 castration resistant metastatic prostate cancer cells (mCRPC) through the hallmarks of apoptosis. The recovery of PC-3 cells from apoptosis upon removal of CBZ at different recovery periods was evaluated by Annexin V, DNA damage, oxidative damage, mitochondrial membrane depolarization, and caspase activation. Our results showed that the administration of CBZ caused apoptosis for 72 h in PC-3 cells. However, recovered cells exhibited decreased nuclear damage, plasma membrane disruption, ROS level, release cytochrome c level and caspase-3 activation with upregulation of Bcl-2 expression upon removal of especially 1 nM CBZ for 24 h recovery period in PC-3 cells. Our study indicates that CBZ treated PC-3 cells can recover after apoptotic cell death. However, advanced molecular analysis should elucidate the relationship between the molecular mechanisms of recovery and taxane response or resistance in PC-3 mCRPC cells.

Promoter hypermethylation as a novel regulator of ANO1 expression and function in prostate cancer bone metastasis

Despite growing evidence implicating the calcium-activated chloride channel anoctamin1 (ANO1) in cancer metastasis, its direct impact on the metastatic potential of prostate cancer and the possible significance of epigenetic alteration in this process are not fully understood. Here, we show that ANO1 is minimally expressed in LNCap and DU145 prostate cancer cell lines with low metastatic potential but overexpressed in high metastatic PC3 prostate cancer cell line. The treatment of LNCap and DU145 cells with DNMT inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) potentiates ANO1 expression, suggesting that DNA methylation is one of the mechanisms controlling ANO1 expression. Consistent with this notion, hypermethylation was detected at the CpG island of ANO1 promoter region in LNCap and DU145 cells, and 5-Aza-CdR treatment resulted in a drastic demethylation at promoter CpG methylation sites. Upon 5-Aza-CdR treatment, metastatic indexes, such as cell motility, invasion, and metastasis-related gene expression, were significantly altered in LNCap and DU145 cells. These 5-Aza-CdR-induced metastatic hallmarks were, however, almost completely ablated by stable knockdown of ANO1. These in vitro discoveries were further supported by our in vivo observation that ANO1 expression in xenograft mouse models enhances the metastatic dissemination of prostate cancer cells into tibial bone and the development of osteolytic lesions. Collectively, our results help elucidate the critical role of ANO1 expression in prostate cancer bone metastases, which is epigenetically modulated by promoter CpG methylation.

CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is associated with resistance to androgen deprivation therapy, and an increase in the population of neuroendocrine (NE) differentiated cells. It is hypothesized that NE differentiated cells secrete neuropeptides that support androgen-independent tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. The cytochrome b561 (CYB561) gene, which codes for a secretory vesicle transmembrane protein, is constitutively expressed in NE cells and highly expressed in CRPC. CYB561 is involved in the α-amidation-dependent activation of neuropeptides, and contributes to regulating iron metabolism which is often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression and maintenance of the highly aggressive NE phenotype in CRPC. In our study, we found that CYB561 expression is upregulated in metastatic and NE prostate cancer (NEPC) tumors and cell lines compared to normal prostate epithelia, and that its expression is independent of androgen regulation. Knockdown of CYB561 in androgen-deprived LNCaP cells dampened NE differentiation potential and transdifferentiation-induced increase in iron levels. In NEPC PC-3 cells, depletion of CYB561 reduced the secretion of growth-promoting factors, lowered intracellular ferrous iron concentration, and mitigated the highly aggressive nature of these cells in complementary assays for cancer hallmarks. These findings demonstrate the role of CYB561 in facilitating transdifferentiation and maintenance of NE phenotype in CRPC through its involvement in neuropeptide biosynthesis and iron metabolism pathways.

Dickkopf-1 (DKK1) drives growth and metastases in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is associated with a poor prognosis and remains an incurable fatal disease. Therefore, the identification of molecular markers involved in cancer progression is urgently needed to develop more-effective therapies. The present study investigated the role of the Wnt signaling modulator Dickkopf-1 (DKK1) in the growth and metastatic progression of mCRPC. DKK1 silencing through siRNA and deletion via CRISPR/Cas9 editing were performed in two different metastatic castration-resistant prostate cancer cell lines (PC3 and DU145). A xenograft tumor model was used to assess tumor growth and metastases. In in vitro experiments, both DKK1 silencing and deletion reduced cell growth and migration of both cell lines. DKK1 knockout clones (DKK1-KO) exhibited cell cycle arrest, tubulin reorganization, and modulation of tumor metastasis-associated genes. Furthermore, in DKK1-KO cells, E-cadherin re-expression and its membrane co-localization with β-catenin were observed, contributing to reduced migration; Cadherin-11, known to increase during epithelial-mesenchymal transition, was down-regulated in DKK1-KO cells. In the xenograft mouse model, DKK1 deletion not only reduced tumor growth but also inhibited the formation of lung metastases. In conclusion, our findings support the key role of DKK1 in the growth and metastatic dissemination of mCRPC, both in vitro and in vivo.

An efficient methodological approach for synthesis of selenopyridines: generation, reactions, anticancer activity, EGFR inhibitory activity and molecular docking studies.

In the present work, we successfully synthesized Se-alkyl selenopyridines 1 and 3, selenopheno[2,3-b]pyridine 2, and bis-selenopyridine 4 derivatives using an eco-friendly method by utilizing NaHSe instead of toxic hydrogen selenide. The effect of the temperature on the reaction was screening at various temperatures. The regiospecific reaction of selenopyridine 1 with bromine afforded an unexpected product 4,6-diamino-5-bromo-2-[(cyanomethyl)selenyl]-pyridine-3-carbonitrile (5), which was cyclized to selenopheno[2,3-b]pyridine (7) by refluxing in the presence of TEA. While its treatment with thiophenol and/or p-chlorothiophenol gave 8a, b. On the other hand, its reaction with aminothiophenol afforded 2-(benzo[d]-thiazol-2-yl)-5-bromoselenopheno[2,3-b]pyridine-3,4,6-triamine (9). Also, N-(2-cyano-4-methyl-5H-1-seleno-3,5,8-triazaacenaphthylen-7-yl)acetamide (11) and a novel series of selenoazo dyes 12a-d were synthesized by treatment of selenopheno[2,3-b]pyridine 2 with acetic anhydride and/or diazonium chlorides of aromatic amines, respectively. Then, we ascertained the potential activity of synthesized compounds against highly metastatic prostate cancer cells (PC-3) and osteosarcoma cells (MG-63) and found that 12a, 12b, 12c, and 12d were more cytotoxic than doxorubicin in both tested cell lines, showing nearly the same anticancer activity with IC50 values ranging from 2.59 ± 0.02µM to 3.93 ± 0.23µM. Mechanistically, the most potent compounds 12a and 12b proved to be potent EGFR inhibitors with IC50 values of 0.301 and 0.123µM, respectively, compared to lapatinib as a positive reference (IC50 = 0.049µM). Moreover, the docking results are in good agreement with the anticancer activity as well as the EGFR inhibitory activity, suggesting these two compounds as promising EGFR anticancer candidates.

Accelerating Cellular Uptake with Unnatural Amino Acid for Inhibiting Immunosuppressive Cancer Cells.

Targeting immunosuppressive metastatic cancer cells is a key challenge in therapy. We recently have shown that a rigid-rod aromatic, pBP-NBD, that responds to enzymes and kill immunosuppressive metastatic osteosarcoma (mOS) and castration resistant prostate cancer (CRPC) cells in mimetic bone microenvironment. However, pBP-NBD demonstrated moderate efficacy against CRPC cells. To enhance activity, we incorporated the unnatural amino acid L- or D-4,4'-biphenylalanine (L- or D-BiP) into pBP-NBD, drastically increasing cellular uptake and CRPC inhibition. Specifically, we inserted BiP into pBP-NBD to target mOS (Saos2 and SJSA1) and CRPC (VCaP and PC3) cells with overexpressed phosphatases. Our results show that the D-peptide backbone with an aspartate methyl diester at the C-terminal offers the highest activity against these immunosuppressive mOS and CRPC cells. Importantly, imaging shows that the peptide assemblies almost instantly enter the cells and accumulate primarily within the endoplasmic reticulum of Saos2, SJSA1, and PC3 cells and at the lysosomes of VCaP cells. By using BiP to boost cellular uptake and self-assembly within cancer cells, this work illustrates an unnatural hydrophobic amino acid as a versatile and effective residue to boost endocytosis of synthetic peptides for intracellular self-assembly.

Abstract 4656: Novel mitochondria-based targeting overcomes resistance in prostate cancer

Abstract Background: Prostate cancer (PC) is a major contributor to cancer-related mortality in men, with metastatic castration-resistant PC (mCRPC) presenting a significant obstacle due to its resistance to standard treatments. Current therapies, including nonsteroidal antiandrogens and chemotherapy drugs, provide limited effectiveness and are often accompanied by severe side effects. Recently, statins have gained attention as antitumor agents, demonstrating chemopreventive effects on a range of human cancers while maintaining a well-established safety profile. However, the use of statins as standalone treatment is limited by insufficient cancer cell targeting. Methods: We synthesized a DZ-simvastatin (DZ-SIM) conjugate that combines a near-infrared heptamethine carbocyanine dye (DZ) with simvastatin for selective tumor cell targeting. Cancer cell survival was measured by crystal violet and MTT assay. Co-localization of DZ-SIM with mitochondria and lysosome in cells was detected under confocal microscope using Mitotracker and Lysotracker. Cellular and mitochondrial cholesterol levels were detected by LC-MS. Male nude mice were inoculated subcutaneously with 22Rv1-Luc cells as a prostate cancer xenograft mouse model. We used Swiss-PDB and Rosetta algorithms to model the structure of the OATP1B3 channel. Oxygen consumption rate and extracellular acidification rate were measured by Seahorse assay. After treatment with DZ-SIM, differentially expressed genes profiling of prostate cancer cells was performed using RNAseq. Results: Our research demonstrates the conjugate's ability to effectively kill androgen-independent and metastatic PC cells without harming normal cells. Furthermore, DZ-SIM has been shown to kill multiple human PC cell lines, regardless of androgen receptor status or therapeutic resistance. DZ-SIM has been shown to inhibit growth in human prostate cancer cell line xenograft models. NIR fluorescence imaging showed specific localization of DZ-SIM to cancer, but not to normal cells and tissues. It could localize into cancer cells by binding the transmembrane transporter OATP to primarily target subcellular organelle mitochondria, decreasing the OCR and ECAR of cancer cells. DZ-SIM can bind to multiple cytosolic and mitochondrial proteins as detected by NIRF imaging. Cholesterol levels also decrease in whole cells and mitochondria. Ingenuity Pathway Analysis (IPA) of RNAseq showed that the four signaling pathways in the DZ-SIM treatment group are significantly enriched in genes related to cholesterol synthesis. Conclusion: DZ-SIM specifically targets PC cells and their mitochondria, resulting in loss of mitochondrial function and consequent cell death. With a unique subcellular targeting strategy, the conjugate holds the potential to outperform existing chemotherapeutic drugs. Citation Format: Yan Ou, Adrian Lim, Ning Zhai, Mouad Edderkaoui, Stephen Pandol, Ruoxiang Wang, Yi Zhang. Novel mitochondria-based targeting overcomes resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4656.

Abstract 2036: Characterizing PARP inhibitor and next-generation anti-androgen therapy responses in advanced prostate cancer

Abstract PARP inhibitors (PARPi) have been shown to be effective in prostate cancer patients with certain DNA repair defects, thus ushering in the era of precision medicine for this disease. Despite this advance, barriers remain which impede further progress including an incomplete understanding of therapeutic response. Our previous and on-going work shows that PARP inhibition leads to both cell death and a more cytostatic, senescence-like arrest. We hypothesize that persistent cells may later promote progression and treatment resistance. Recent clinical trials such as PROpel and TALAPRO-2 have demonstrated success in combining PARPi’s with next-generation anti-androgen therapies (NGAT) including abiraterone and enzalutamide. Whether NGAT’s are synthetic lethal and cell death promoting with PARP inhibition is poorly understood. Additionally, given the evolving clinical landscape for prostate cancer, it is important to test whether prior NGAT exposure may preclude response to combination therapy. In this study, we sought to characterize these combinations to better understand tumor cell response to treatment and the placement of treatment within the overall therapeutic paradigm. We’ve previously used the C4-2B metastatic castration-resistant prostate cancer cell line as a model which responds well to PARP inhibition. Here, we additionally utilize the C4-2B-derived enzalutamide and abiraterone resistant lines MDVR and AbiR, respectively. The combination of olaparib with abiraterone (recently approved from results of PROpel) was significantly more efficacious than either monotherapy in all three models. Additionally, combination of talazoparib and enzalutamide (approved from results of TALAPRO-2) was also more effective than monotherapy. These data suggest that prior development of NGAT resistance does not prevent benefit from NGAT and PARPi combinations. Although both combinations were found to cause significantly less cell growth, cells appeared more cytostatic rather than apoptotic, suggesting NGAT’s do not induce significant synthetic lethality with PARPi’s. Our work suggests that combining a PARPi with an NGAT promotes a persistent cytostasis which may give rise to eventual tumor progression and the development of resistance. Future studies are directed at further characterizing combinations with PARPi’s and the development of novel strategies to enhance their efficacy. Citation Format: Love A. Moore, Bryan Correa Gonzalez, Allen C. Gao, Alan P. Lombard. Characterizing PARP inhibitor and next-generation anti-androgen therapy responses in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2036.

Abstract 7200: Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy

Abstract Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, most men succumb to Metastatic Castration Resistant Prostate Cancer (mCRPC) due to treatment resistance. MAGMAS, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. MAGMAS is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, MAGMAS plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of MAGMAS in treatment-resistant PCa cells. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of MAGMAS compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX-resistant metastatic PCa cell lines and re-sensitizes them to DTX. MAGMAS knockdown in DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of MAGMAS with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using cell migration/invasion assays and tumor-sphere formation assays are underway to further establish MAGMAS as a potential therapeutic target to attenuate the aggressive properties of chemo-resistant PCa cells. Lastly, we will investigate the diagnostic potential of MAGMAS as a biomarker for advanced PCa and BT#9 as an imaging/therapeutic-specific agent for treatment-resistant tumors. Citation Format: Alfonso Duran, Kristen Whitley, Krystal Santiago, Adil Mohammed, Carlos Casiano, Bhaskar Das, Frankis Almaguel. Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7200.

Abstract 560: IGF1 induces the expression of CYR61 in metastatic prostate cancer

Abstract The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. Interestingly, CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Our goal was to assess the contribution of CYR61-IGF1 interaction to PCa cell proliferation. Their combined role may explain a proportion of aggressive PCa. This study was designed to characterize the role of IGF1-induced expression of CYR61 in metastatic PCa cellular models and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, migration, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. In addition, we found that IGF1 induces protein expression of CYR61 in a time-dependent manner, validated by both immunoblotting and confocal microscopy. In conclusion, these data provided evidence that CYR61 expression is induced by IGF1, and its inhibition decreased the capacity of metastatic PCa cells to proliferate, indicating a potential utilization of CYR61 inhibition in the therapy of metastatic PCa. Citation Format: Greisha L. Ortiz Hernandez, Mya Walker, Leanne Woods-Burnham, Rick A. Kittles, Carlos A. Casiano, Susan Neuhausen. IGF1 induces the expression of CYR61 in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 560.

Abstract 161: Aged mice decelerate bone metastasis of prostate cancer by decreasing infiltration of M2 macrophages

Abstract Bone metastasis is a major contributor to the mortality of patients with prostate cancer and results in the development of an immunosuppressive microenvironment in the bone marrow. While advanced age increases the risk of bone metastasis, effects of age-related changes on immune microenvironmental homeostasis are not yet clearly understood. To investigate interactions between cancer cells and the immune microenvironment in elderly individuals, we utilized murine-derived specific bone metastatic prostate cancer cells (RM1-BM), which we had previously isolated from bone metastatic mice and transfected with a luciferase-based reporter gene. We then injected the cells into the left ventricle of both 3‒6 months and 22‒24 months, male, wild-type mice to induce bone metastasis, using age-matched mice injected with PBS as a control. We employed in vivo imaging to compare the metastatic levels in both groups of mice every other day. On day 14, we extracted bone marrow cells for CyTOF analysis and femurs and tibias for Hematoxylin and Eosin staining. Interestingly, our preliminary results demonstrated that healthy aged mice exhibited a lower susceptibility to develop bone metastasis when compared to their younger counterparts. A further investigation of the immune subtypes revealed an increase in the percentage of macrophages in CD45+ immune cells in young mice that developed bone metastasis as opposed to age-matched controls. However, the percentage decreased in aged mice, going from 12.20% to 9.92% (p = 0.001). Lastly, macrophages were isolated from the bone marrow of 3‒6 months and 22‒24 months old mice and were co-cultured with ovalbumin-transfected RM1-BM cells (RM1-BM-ova) in vitro for 24 hours. The flow cytometry results indicated that both groups consist mainly of CD206+ (M2) macrophages. Nonetheless, those obtained from aged mice exhibited reduced ovalbumin presentation capability while maintaining a steady level of type I major histocompatibility complex (MHC-I). These findings suggested that increased infiltration of pro-inflammatory M2 macrophages within the tumor microenvironment may contribute to the bone metastasis of prostate cancer. Therefore, inhibiting M2 macrophages recruitment to the bone marrow microenvironment may prove a useful approach for treating patients with prostate cancer bone metastasis. This study was supported by the NSFC projects (81972420, 81972766, 82173336), and grants from the Shenzhen Science and Technology Innovation Commission (JCYJ20190809161811237, JCYJ20210324104214040). Citation Format: Xiao Zhang, Wei Yu, Xin Huang, Yi Lu, Jian Zhang. Aged mice decelerate bone metastasis of prostate cancer by decreasing infiltration of M2 macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 161.

Effects of metformin and silodosin as supplementary treatments to abiraterone on human telomerase reverse transcriptase (hTERT) level in metastatic castration-resistant prostate cancer (mCRPC) cells: An in vitro study.

The antiproliferative properties of metformin and silodosin have been observed in prostate cancer. Furthermore, it is hypothesized that the molecular pathways related to these drugs may impact the levels of human telomerase reverse transcriptase (hTERT) in prostate cancer cells. The aim of this study was to assess the effect of metformin and silodosin on the levels of hTERT in metastatic castration-resistant prostate cancer (mCRPC) cells. The present study employed an experimental design with a post-test-only control group. This study utilized the PC3 cell line as a model for mCRPC. A viability experiment was conducted using the CCK-8 method to determine the inhibitory concentration (IC50) values of metformin, silodosin, and abiraterone acetate (AA) after a 72-hour incubation period of PC3 cells. In order to investigate the levels of hTERT, PC3 cells were divided into two control groups: a negative control and a standard therapy with AA. Additionally, three experimental combination groups were added: metformin with AA; silodosin with AA; and metformin, silodosin and AA. The level of hTERT was measured using sandwich ELISA technique. The difference in hTERT levels was assessed using ANOVA followed by a post hoc test. The IC50 values for metformin, silodosin, and AA were 17.7 mM, 44.162 mM, and 66.9 μM, respectively. Our data indicated that the combination of metformin with AA and the combination of metformin, silodosin and AA decreased the hTERT levels when compared to control, AA, and silodosin with AA. The administration of metformin resulted in a reduction of hTERT levels in the PC3 cell line, but the impact of silodosin on hTERT levels was not statistically significant compared to AA group.

Tramadol and/or Ketamine Repurposing as Potential Anticancer Drugs in Metastatic Castration-Resistant Prostate Cancer Cell Lines

Tramadol and/or Ketamine Repurposing as Potential Anticancer Drugs in Metastatic Castration-Resistant Prostate Cancer Cell Lines

Radiotherapy Metastatic Prostate Cancer Cell Lines Treated with Gold Nanorods Modulate miRNA Signatures.

MicroRNA (miRNA) modulation has been identified as a promising strategy for improving the response of human prostate cancer (PCa) to radiotherapy (RT). Studies have shown that mimics or inhibitors of miRNAs could modulate the sensitivity of PCa cells to RT. In addition, pegylated gold nanoparticles have been studied as a therapeutic approach to treat PCa cells and/or vehicles for carrying miRNAs to the inside of cells. Therefore, we evaluated the capacity of hypofractionated RT and pegylated gold nanorods (AuNPr-PEG) to modulate the miRNA signature on PCa cells. Thus, RT-qPCR was used to analyze miRNA-95, miRNA-106-5p, miRNA-145-5p, and miRNA-541-3p on three human metastatic prostate cell lines (PC3, DU145, and LNCaP) and one human prostate epithelial cell line (HprEpiC, a non-tumor cell line) with and without treatment. Our results showed that miRNA expression levels depend on cell type and the treatment combination applied using RT and AuNPr-PEG. In addition, cells pre-treated with AuNPr-PEG and submitted to 2.5 Gy per day for 3 days decreased the expression levels of miRNA-95, miRNA-106, miRNA-145, and miRNA-541-3p. In conclusion, PCa patients submitted to hypofractionated RT could receive personalized treatment based on their metastatic cellular miRNA signature, and AuNPr-PEG could be used to increase metastatic cell radiosensitivity.

End-of-Life Care Preferences of Patients with Advanced Urological Malignancies: An Explorative Survey Study at a Tertiary Referral Center.

Background: Many people want to die at home, but it is often not possible because they do not share their wishes with family members. This study was conducted to find out the extent to which patients with advanced urological malignancies had wishes regarding their final stage of life, made arrangements accordingly, and communicated their wishes to relatives and health care professionals. Methods: We conducted a survey among advanced urological tumor patients during their clinic visit at a German university hospital using a 31-item questionnaire. Inclusion criteria were metastatic or irresectable prostate cancer, urothelial carcinoma, or renal cell carcinoma. Results: In total, 88 patients (76 male, 12 female) completed the questionnaire, and 62 of those respondents (70%) had received their tumor diagnosis within the past 5 years. Symptoms were reported by 80%, and 18% described five or more symptoms. The majority (88%) stated that they had thought about their preferred place of death but 58% had not informed anyone about it. The preference for a hospice as the place of death correlated statistically significantly with the absence of a domestic partnership (p = 0.001) or marriage (p < 0.001) and with a high number of symptoms (≥5; p = 0.009). However, 73% had not talked with their urological oncologist about care options in case their health deteriorated though 36% of those were interested in having a conversation about it. Conclusions: Our results showed that 9 out of 10 patients reflected on their preferred place of death but only a few discussed it with anyone. Based on this finding, physicians and healthcare staff should initiate discussions about early care planning so that patients in incurable situations can express their wishes regarding their preferred place of death.

Quality by design fostered fabrication of cabazitaxel loaded pH-responsive Improved nanotherapeutics against prostate cancer

Cabazitaxel has been approved for the treatment of prostate cancer since 2010. However, its poor solubility and permeability pitfalls prevent its accumulation at the target site and promote severe adverse effects. About 90% of prostate cancer (PCa) patients suffer from bone metastasis. This advent reports the development of CBZ-loaded pH-responsive polydopamine nanoparticles (CBZ NP) against metastatic PCa cells. Quality by design (QbD) and multivariate analysis tools were employed for the optimization of CBZ NP. Amorphisation of CBZ along with metastatic microenvironment responsive release was observed thereby imparting spatial release and circumventing solubility pitfalls. CBZ NP retained its cytotoxic potential, with a significant increase in quantitative cellular uptake. Apoptotic markers observed from nuclear staining with elevated reactive oxygen species (ROS) and mitochondrial damage revealed by JC-1 staining demonstrated the efficacy of CBZ NP against PC-3 cells with good serum stability and diminished hemolysis. Cell cycle analysis revealed substantial S and G2/M phase arrest with enhancement in apoptosis was observed. Western blot studies revealed an elevation in caspase-1 and suppression in Bcl-2 indicating enhanced apoptosis compared to the control group. Substantial reduction in the diameter of 3D-Tumoroid and enhanced cell proliferation inhibition indicated the efficacy of CBZ NP in PCa. Thus, we conclude that CBZ NP could be a promising Nanotherapeutic approach for PCa.