Background SGN-B7H4V is a novel investigational vedotin antibody–drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex.Methods B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model.Results Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent.Conclusion The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.

PD-L1 (22C3) checkpoint inhibitor therapy represents a mainstay of modern cancer immunotherapy for non-small cell lung cancer (NSCLC). In vitro diagnostic (IVD) PD-L1 antibody staining is widely used to predict clinical intervention efficacy. However, pathologist interpretation of this assay is cumbersome and variable, resulting in poor positive predictive value concerning patient therapy response. To address this, we developed a digital assay (DA) termed Tissue Insight (TI) 22C3 NSCLC, for the quantification of PD-L1 in NSCLC tissues, including digital recognition of macrophages and lymphocytes. We completed clinical validation of this digital image analysis solution in 66 NSCLC patient samples, followed by concordance studies (comparison of PD-L1 manual and digital scores) in an additional 99 patient samples. We then combined this DA with three distinct immune cell recognition algorithms for detecting tissue macrophages, alveolar macrophages, and lymphocytes to aid in sample interpretation. Our PD-L1 (22C3) DA was successfully validated and had a scoring agreement (digital to manual) higher than the inter-pathologist scoring. Furthermore, the number of algorithm-identified immune cells showed significant correlation when compared with those identified by immunohistochemistry in serial sections stained by double immunofluorescence. Here, we demonstrated that TI 22C3 NSCLC DA yields comparable results to pathologist interpretation while eliminating the intra- and inter-pathologist variability associated with manual scoring while providing characterization of the immune microenvironment, which can aid in clinical treatment decisions.

Background: Immune cells within the tumor microenvironment (TME) play a vital role in regulating tumor progression. Therefore, immunotherapies that elicit anti-tumor responses are of great interest for the treatment of various cancers. Macrophages are a current immune cell type of interest due to the ability to polarize into anti-tumor (M1) and pro-tumor (M2) phenotypes. The density and phenotype of macrophages within the tumor and TME have been linked to prognosis in multiple types of solid tumors. Our hypothesis is that colorectal cancer (CRC) patients with high immune infiltration and greater amounts of anti-tumor immune cells within the tumor compartment will have an increased time of survival compared to cancers with immune excluded or desert environments. Methods: One CRC tumor microarray (TMA) containing primary tumors, metastases, and normal tissue were stained via multiplex immunofluorescence (mIF) for 6 different immune markers: CD3, CD8, CD56, CD68, CD163, and PD-L1. The stained TMAs were analyzed utilizing Flagship Biosciences’ proprietary image analysis platform. Machine learning algorithms used cellular features to stratify cells as belonging to either the tumoral or stromal compartment. Core level expression data was pulled and represented on a whole-cohort basis. All staining and image analysis outputs were reviewed by a board-certified, MD pathologist. Kaplan-Meier curves were generated using survival data in relation to the low, medium, and high expression of CD68, CD163, and/or PDL1 in the whole tissue, as well as tumor and stromal compartments. Patients were also stratified into low (1 and 2) and high (3 and 4) cancer stages. Results: There is a correlation between patient survival and the presence or absence of macrophage markers CD68 and CD163 and lack of immune-suppressive marker PDL1. Specifically, lower levels of CD68+CD163- cells within the tumor compartment correlate with an increase in patient survival. Similarly, lower PDL1 expression in the stromal compartment positively correlate with prolonged patient survival in CRC patients. Conclusion: Data generated through Flagship Biosciences’ image analysis platform showed a strong relationship between CD68 and CD163 presence and localization with CRC patient survival. Altering the immune cells within the tumor to an anti-tumor immune environment could increase patient survival times in high stage CRC patient populations. Combining immune checkpoint inhibitors with current FDA approved therapies for CRC are of interest to further extend patient survival, as PDL1 expression negatively correlate with CRC patient survival. Further, utilizing Flagship Biosciences’ image analysis software to understand cancer immune microenvironments should be further utilized to aid in diagnosis and treatment decisions. Citation Format: Dannah Miller, Kate Hieber, Will Paces, Huong Nguyen, Adam Beharry, Roberto Gianani. Macrophage biomarkers CD68 and CD163 correlate with CRC patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2530.

The castration-resistant (CR) prostate cancer (PCa) is lethal and is the second leading cause of cancer-related deaths in U.S. males. To develop effective treatments toward CR PCa, we investigated reactive oxygen species (ROS) signaling pathway for its role involving in CR PCa progression. ROS can regulate both cell growth and apoptosis: a moderate increase of ROS promotes proliferation; its substantial rise results in cell death. p66Shc protein can increase oxidant species production and its elevated level is associated with the androgen-independent (AI) phenotype of CR PCa cells; while heme oxygenase-1 (HO-1) is an antioxidant enzyme and elevated in a sub-group of metastatic PCa cells. In this study, our data revealed that HO-1 and p66Shc protein levels are co-elevated in various AI PCa cell lines as well as p66Shc cDNA-transfected cells. Knockdown and/or inhibition of either p66Shc or HO-1 protein leads to reduced tumorigenicity as well as a reduction of counterpart protein. Knockdown of HO-1 alone results in increased ROS levels, nucleotide and protein oxidation and induction of cell death. Together, our data indicate that elevated HO-1 protein levels protect PCa cells from otherwise apoptotic conditions induced by aberrant p66Shc/ROS production, which thereby promotes PCa progression to the CR phenotype. p66Shc and HO-1 can serve as functional targets for treating CR PCa.

Prostate cancer is the most commonly diagnosed solid tumor and the second leading cause of cancer-related deaths in men in the United States. While localized prostate cancer has an excellent prognosis for patients, about one-third of patients are diagnosed with high-risk disease, including metastatic cancer. The 5-year survival rate of metastatic prostate cancer is only about 30%. Due to the androgen dependence of prostate cancer cells, androgen-deprivation therapy is the standard of care for metastatic prostate cancer, which includes both surgical and medical approaches. Nevertheless, androgen-deprivation therapy in general is not curative; patients can develop castration-resistant prostate cancer. Despite current chemotherapies, including the utilization of novel androgen signaling inhibitors and immunotherapy, patients still succumb to the disease. Hence, castration-resistant prostate cancer is a lethal disease. Combination treatment is a strategy for treating this lethal disease and thus will be the focus of discussion in this chapter.

BackgroundImmune cells within the tumor microenvironment (TME) play a vital role in regulating tumor progression. Therefore, immunotherapies that stimulate anti-tumor responses are of great interest for the treatment of various cancers. PD-L1 expression on immune cells is positively correlated with increased patient survival. Our hypothesis is that non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) patients with high immune infiltration and greater amounts of anti-tumor immune cells within the tumor compartment have an increased time of survival compared to cancers with immune excluded or immune desert environments.MethodsOne NSCLC and one CRC tumor microarray (TMA) containing primary tumors, metastases, and normal tissue were stained via multiplex immunofluorescence (mIF) for 6 different immune markers: CD3, CD8, CD56, CD68, CD163, and PD-L1. This multiplex panel was designed to evaluate the immune cell population as well as tumor and immune cell PD-L1 status to aid in research for immunotherapies, specifically anti-PD-L1 therapies. The stained TMAs were analyzed utilizing Flagship Biosciences’ proprietary image analysis platform. Machine learning algorithms stratified cells as belonging to the tumoral or stromal space based on their cellular features. Core level expression data was pulled and represented on a whole-cohort basis. All staining and image analysis outputs were reviewed by a board-certified, MD pathologist. Kaplan-meier curves were generated based on survival data in relation to the levels of immune cells present within the tumor cores as well as the percentage of immune cells infiltrating into the tumor.ResultsThere is a clear correlation between patient survival and the presence or absence of various types of immune cells, including helper T cells, cytotoxic T cells, M1 macrophages, M2, macrophages, NK cells, as well as PDL1 expression on tumor and immune cells. Specifically, the increased presence of anti-tumor immune cells as well as increased expression of PD-L1 on immune cells within the tumor compartment correlates with an increase in patient survival.ConclusionsData generated through Flagship Biosciences’ image analysis platform showed a strong relationship between immune cell presence and localization and NSCLC and CRC patient survival. Altering the immune cells within the tumor to an anti-tumor immune environment could increase patient survival times. Combining immune checkpoint inhibitors with current FDA approved therapies for NSCLC and CRC are of interest to further extend patient survival. Further, utilizing Flagship Biosciences’ image analysis software to understand cancer immune microenvironments should be further utilized to aid in diagnosis and treatment decisions.

Beginning with the earliest studies of autophagy in cancer, there have been indications that autophagy can both promote and inhibit cancer growth and progression; autophagy regulation of organelle homeostasis is similarly complicated. In this review we discuss pro- and antitumor effects of organelle-targeted autophagy and how this contributes to several hallmarks of cancer, such as evading cell death, genomic instability, and altered metabolism. Typically, the removal of damaged or dysfunctional organelles prevents tumor development but can also aid in proliferation or drug resistance in established tumors. By better understanding how organelle-specific autophagy takes place and can be manipulated, it may be possible to go beyond the brute-force approach of trying to manipulate all autophagy in order to improve therapeutic targeting of this process in cancer.

Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cause of cancer-related deaths in U.S. men in 2020. Androgen-deprivation therapy (ADT) is the standard of care for metastatic PCa. Unfortunately, PCa relapse often occurs one to two years after initiation of ADT, resulting in the development of castration-resistant PCa (CRPCa), a lethal disease. While several anticancer agents such as docetaxel, abiraterone acetate, and enzalutamide are currently utilized to extend a patient's life after development of CRPCa, patients will eventually succumb to the disease. Hence, while targeting androgen signaling and utilization of docetaxel remain the most crucial agents for many of these combinations, many studies are attempting to exploit other vulnerabilities of PCa cells, such as inhibition of key survival proteins, anti-angiogenesis agents, and immunotherapies. This review will focus on discussing recent advances on targeting therapy. Several novel small molecules will also be discussed.

BackgroundMALT1 protease is a promising target in aggressive lymphomas1, and two phase 1 clinical trials in hematological cancers are ongoing (NCT03900598, NCT04876092). More recently, MALT1 protease inhibition was also shown to reprogram regulatory T cells (Treg) in solid tumors, causing them to lose their immunosuppressive function and secrete interferon-gamma (IFN).2 Changes in Treg metabolism in the tumor microenvironment (TME) may account for their destabilization and selective susceptibility to reprogramming in tumor tissue.3 4 5 While strong MALT1 inhibition can cause Treg depletion in blood and induce autoimmune toxicity,6 a therapeutic window for a differentiated MALT1 inhibitor that reprograms destabilized Treg in the TME before affecting Treg in healthy tissue may exist.2 MPT-0118 is an orally dosed MALT1 inhibitor developed to reprogram destabilized Treg in the TME without causing autoimmune symptoms. A Phase 1/1b dose-escalation and cohort-expansion clinical trial evaluating MPT-0118 is underway (NCT04859777).MethodsHuman xenograft models of lymphoma were used to assess the direct activity of MPT-0118 on MALT1-dependent (but not not MALT1-independent) hematologic tumors. Effects of MPT-0118 on solid tumors were determined in syngeneic cancer models. Human and mouse tumor tissues were evaluated for Treg reprogramming by in situ hybridization or flow cytometry. Patient-derived organotypic tumor spheroids were assessed for immune-mediated cell killing. Studies in rodents and dogs assessed pharmacokinetics (PK) and safety.ResultsMPT-0118 was selective and effective in preventing growth of aggressive MALT1 protease-dependent lymphomas. Beyond direct activity on hematologic malignancies, MPT-0118 also increased anti-tumor immune responses as single-agent or in combination with anti-PD-1 in syngeneic tumor models that are otherwise unresponsive to immune checkpoint blockade (ICB). MPT-0118-treated syngeneic tumors showed an increase in IFN-secreting Treg, associated with decelerated tumor growth. PK studies reveal that MPT-0118 has a high volume of distribution, and effective inhibitor concentrations are reached in the murine tumors upon oral dosing. The drug candidate caused tumor-associated Treg to produce IFN without changing the frequency of Treg circulating in the blood. Ex vivo, MPT-0118 induced Treg reprogramming in tumors resected from patients with colorectal and endometrial cancers and cell killing in spheroids derived from patients with colorectal cancer.ConclusionsThe MALT1 inhibitor MPT-0118 is a clinical candidate for treating MALT1-expressing lymphomas and Treg-infiltrated solid tumors. MPT-0118 exploits the therapeutic opportunity presented by destabilized Treg in the TME. Treg reprogramming represents a novel strategy with the potential to improve responses to ICB therapy in a broad range of solid tumors.ReferencesNagel D, Spranger S, Vincendeau M, Grau M, Raffegerst S, Kloo B, Hlahla D, Neuenschwander M, Peter von Kries J, Hadian K, Dörken B, Lenz P, Lenz G, Schendel DJ, Krappmann D. Pharmacologic inhibition of MALT1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive ABC-DLBCL. Cancer Cell 2012 December 11;22(6):825–37.Di Pilato M, Kim EY, Cadilha BL, Prüßmann JN, Nasrallah MN, Seruggia D, Usmani SM, Misale S, Zappulli V, Carrizosa E, Mani V, Ligorio M, Warner RD, Medoff BD, Marangoni F, Villani AC, Mempel TR. Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy. Nature 2019 June;570(7759):112–116.Lim SA, Wei J, Nguyen TM, Shi H, Su W, Palacios G, Dhungana Y, Chapman NM, Long L, Saravia J, Vogel P, Chi H. Lipid signalling enforces functional specialization of Treg cells in tumours. Nature 2021 March;591(7849):306–311.Zappasodi R, Serganova I, Cohen IJ, Maeda M, Shindo M, Senbabaoglu Y, Watson MJ, Leftin A, Maniyar R, Verma S, Lubin M, Ko M, Mane MM, Zhong H, Liu C, Ghosh A, Abu-Akeel M, Ackerstaff E, Koutcher JA, Ho PC, Delgoffe GM, Blasberg R, Wolchok JD, Merghoub T. CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours. Nature 2021 March;591(7851):652–658.Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, Shuai Y, Normolle DP, Kirkwood JM, Ferris RL, Delgoffe GM, Bruno TC, Workman CJ, Vignali DAA. Interferon-γ drives Treg fragility to promote anti-tumor immunity. Cell 2017 June 1;169(6):1130–1141.e11.Martin K, Junker U, Tritto E, Sutter E, Rubic-Schneider T, Morgan H, Niwa S, Li J, Schlapbach A, Walker D, Bigaud M, Beerli C, Littlewood-Evans A, Rudolph B, Laisney M, Ledieu D, Beltz K, Quancard J, Bornancin F, Zamurovic Ribrioux N, Calzascia T. Pharmacological inhibition of MALT1 protease leads to a progressive IPEX-Like pathology. Front Immunol 2020 April 30;11:745.

Autophagy, the process of macromolecular degradation through the lysosome, has been extensively studied for the past decade or two. Autophagy can regulate cell death, especially apoptosis, through selective degradation of both positive and negative apoptosis regulators. However, multiple other programmed cell death pathways exist. As knowledge of these other types of cell death expand, it has been suggested that they also interact with autophagy. In this review, we discuss the molecular mechanisms that comprise three non-apoptotic forms of cell death (necroptosis, pyroptosis and ferroptosis) focusing on how the autophagy machinery regulates these different cell death mechanisms through (i) its degradative functions, i.e., true autophagy, and (ii) other non-degradative functions of the autophagy machinery such as serving as a signaling scaffold or by participating in other autophagy-independent cellular processes.

New pregnene analogs of N-hydroxamic acid 6, imino-propane hydrazides 7 and 8 as well as the aryl amides 9–11, oxadiazole, pyrazole and sulfinyl analogs 13–15, via the hydrazide analog 5 of methyl ((5-pregnen-3β,17β-diol-15α-yl)thio)propanoate (4) were synthesized. The in vitro cytotoxic activities of selected synthesized steroids against two human prostate cancer cell lines (PC-3, and LNCaP-AI) were evaluated by MTT assay. Compound 10 was the most active cytotoxic agent among these steroids against PC-3 and LNCaP-AI cell lines with inhibition of 96.2%, and 93.6% at concentration levels of 10.0 μM and 91.8%, and of 79.8% at concentration of 1.0 μM, respectively. Molecular docking study of 10 showed a hydrogen bonding with the amino acid Asn705 residue of the receptor 1E3G, together with hydrophobic interactions. Therefore, compound 10 can be considered as a promising anticancer agent due to its potent cytotoxic activity.

Currently, prostate cancer (PCa) remains the most commonly diagnosed solid tumor and the second leading cause of cancer-related deaths in US men. Most of these deaths are attributed to the development of castration-resistant (CR) PCa. ErbB-2 and ErbB family members have been demonstrated to contribute to the progression of this lethal disease. In this review, we focus on updating the role of ErbB-2 in advanced PCa progression and its regulation, including its regulation via ligand activation, miRNAs and protein phosphorylation. We also discuss its downstream signaling pathways, including AKT, ERK1/2 and STATs, involved in advanced PCa progression. Additionally, we evaluate the potential of ErbB-2, focusing on its protein hyper-phosphorylation status, as a biomarker for aggressive PCa as well as the effectiveness of ErbB-2 as a target for the treatment of CR PCa via a multitude of approaches, including orally available inhibitors, intratumoral expression of cPAcP, vaccination and immunotherapy.

Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in U.S. men due to the development of the castration-resistant (CR) PCa phenotype. A useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies toward CR PCa. In this study, we established a PCa cell progressive model in three separate cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells. Those AI PCa cells obtain the biochemical properties of the clinical CR phenotype, including AR and PSA expression as well as enhanced proliferation and tumorigenicity under androgen-deprived conditions. Thus, those AI cells recapitulate CR PCa and exhibit increased oxidant species levels as well as enhanced signaling of proliferation and survival pathways. H2O2 treatment directly enhanced AS cell growth and migration, which was counteracted by antioxidant N-acetyl cysteine (NAC). We further identified p66Shc protein enhances the production of oxidant species which contributes to phenotypic and cell signaling alterations from AS to AI PCa cells. H2O2-treated LNCaP-AS cells had a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Conversely, the oxidant species-driven alterations of LNCaP-AI and p66Shc subclone cell signaling is mitigated by p66Shc knockdown. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules, correlating with p66Shc protein levels. Together, the data shows that p66Shc enhances oxidant species production that plays a role in promoting PCa progression to the CR stage.

Purpose and Background: Prostate cancer (PCa) is the second leading cause of cancer-related death in United States men. Androgen deprivation therapy is the standard-of-care treatment for metastatic PCa; most patients eventually relapse and develop castration-resistant (CR) tumors that currently have no effective treatment. Thus, a useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies for CR PCa. In this study, we established a PCa cell progressive model in three independent cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells, recapitulating clinical PCa progression. Experimental Methods: AS and AI human prostate adenocarcinoma cell lines LNCaP, MDA PCa2b and VCaP were utilized in this study to demonstrate the molecular and signaling alterations seen in CR PCa. Stable p66Shc cDNA transfected subclones were established from LNCaP-AS cells. The tumorigenicity of AS and AI cells as well as AS PCa cells treated with H2O2 and NAC were evaluated via trypan blue exclusion, transwell migration, clonogenic assays and xenograft mouse models. The signaling profiles including phosphoprotein microarray and immunoblotting were conducted. Results: AI PCa cells have enhanced tumorigenicity, recapitulating the clinical CR phenotype, including AR expression, proliferation and tumorigenicity under androgen-deprived conditions. Further, AI cells exhibit increased ROS levels as well as enhanced signaling of proliferation and survival pathways. We further identified oxidase p66Shc as one of the potential sources of the ROS-mediated phenotypic and cell signaling alterations in AI PCa cells. LNCaP-AI cells and p66Shc subclones have a greater oxidative environment compared to LNCaP-AS cells. Increased ROS via H2O2 enhanced AS cell growth and migration, which was counteracted by antioxidant NAC. Treatment of LNCaP-AS cells with H2O2 resulted in a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Further, the ROS-driven alterations of p66Shc subclone cell signaling can be mitigated via p66Shc knockdown or inactive p66Shc mutant. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules. Molecular profiling showed that alterations of signaling in LNCaP-AI cells and p66Shc subclones attributed to p66Shc/ROS. Conclusions: We report the establishment of a PCa cell progression model in three commonly used PCa cell lines that replicates clinical PCa progression from the AS to the AI/CR phenotype. Altogether, the data shows ROS produced by p66Shc promotes PCa tumorigenicity and progression to the CR phenotype. Further characterization of the PCa progressive model will aid in the understanding of advanced PCa progression to help in treatment of this lethal disease. Citation Format: Dannah Miller, Matthew Ingersoll, Arpita Chatterjee, Rebecca Oberley-Deegan, Ming-Fong Lin. p66Shc/ROS enhances the progression of androgen-sensitive towards castration-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4285.

The standard-of-care treatment for metastatic prostate cancer (PCa) is androgen deprivation therapy (ADT). Nevertheless, most tumors eventually relapse and develop into lethal castration-resistant prostate cancer (CRPC). Docetaxel is a FDA-approved agent for the treatment of CRPC; however, the tumor often quickly develops resistance to this drug. Thus, there is an immediate need for novel therapies to treat docetaxel-resistant PCa. In this study, we modified the structure of CIL-102 and investigated the efficacy of the derivatives against CRPC and docetaxel-resistant PCa. These novel CIL-102 derivatives inhibit CRPC tumorigenicity, including proliferation, migration and colony formation, and importantly, selectively inhibit CRPC cell proliferation over non-cancerous prostate epithelia. Computational modeling indicated the derivatives bind to β-tubulin and immunocytochemistry revealed the depolymerization of microtubules upon treatment. Western blot analyses reveal that pro-apoptotic and anti-oxidant pathways are activated, and MitoSOX and DCF-DA analyses confirmed increased reactive oxygen species (ROS) production upon treatments. Furthermore, CIL-102 derivatives effectively reduce the proliferation of docetaxel-resistant CR PCa cell lines. Our data indicate the potential of these compounds as promising therapeutic agents for CRPC as well as docetaxel-resistant CRPC.

Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.

The treatment of castration-resistant (CR) prostate cancer (PCa) is limited. A sub-population of CR PCa tumors can synthesize androgens for intracrine androgen receptor (AR) activation, thus targeting androgen biosynthesis could be an effective therapeutic option for these patients. We determined that androgen biosynthesis inhibitors simvastatin, atorvastatin, and ketoconazole directly inhibit growth, migration, and colony formation of LNCaP C-81 cells, which exhibit de novo androgen biosynthesis, with simvastatin being the most effective. Importantly, in combination treatments, statins specifically enhanced growth suppression with added effects by anti-androgen abiraterone acetate on the CR PCa cells. Thus, statins can be used in conjunction with abiraterone acetate to enhance anti-androgen therapy for CR PCa.

Despite recent advances in modern medicine, castration-resistant prostate cancer remains an incurable disease. Subpopulations of prostate cancer cells develop castration-resistance by obtaining the complete steroidogenic ability to synthesize androgens from cholesterol. Statin derivatives, such as simvastatin, inhibit cholesterol biosynthesis and may reduce prostate cancer incidence as well as progression to advanced, metastatic phenotype. In this study, we demonstrate novel simvastatin-related molecules SVA, AM1, and AM2 suppress the tumorigenicity of prostate cancer cell lines including androgen receptor-positive LNCaP C-81 and VCaP as well as androgen receptor-negative PC-3 and DU145. This is achieved through inhibition of cell proliferation, colony formation, and migration as well as induction of S-phase cell-cycle arrest and apoptosis. While the compounds effectively block androgen receptor signaling, their mechanism of inhibition also includes suppression of the AKT pathway, in part, through disruption of the plasma membrane. SVA also possess an added effect on cell growth inhibition when combined with docetaxel. In summary, of the compounds studied, SVA is the most potent inhibitor of prostate cancer cell tumorigenicity, demonstrating its potential as a promising therapeutic agent for castration-resistant prostate cancer.