Moving toward improved immune checkpoint immunotherapy for advanced prostate cancer.

Human prostate cancer is a heterogenous malignancy that responds poorly to immunotherapy targeting immune checkpoints. The immunosuppressive tumor microenvironment that is typical of human prostate cancer has been the main obstacle to these treatments. The effectiveness of these therapies is also hindered by acquired resistance, leading to slow progress in prostate cancer immunotherapy. Results from the highly anticipated late-stage clinical trials of PD-1/PD-L1 immune checkpoint blockade in patients with advanced prostate cancer have highlighted some of the obstacles to immunotherapy. Despite the setbacks, there is much that has been learned about the mechanisms that drive resistance, and new strategies are being developed and tested. Here, we review the status of immune checkpoint blockade and the immunosuppressive tumor microenvironment and discuss factors contributing to innate and adaptive resistance to immune checkpoint blockade within the context of prostate cancer. We then examine current strategies aiming to overcome these challenges as well as prospects.

Intestinal Microbiota and Gene Expression Reveal Similarity and Dissimilarity Between Immune-Mediated Colitis and Ulcerative Colitis.

Immune checkpoint inhibitors (ICIs) have become the standard of care for several cancers. However, ICI therapy has also been associated with various immune-related adverse events (irAEs). Clinical manifestations of immune-related colitis resemble those of inflammatory bowel diseases such as ulcerative colitis (UC). The composition of the bowel microflora is thought to influence the development of inflammatory bowel disease and irAE colitis. We profiled the gene expressions and microbe compositions of colonic mucosa from patients with solid cancers receiving anti-PD-L1 antibody treatment; we then compared the expression profiles associated with irAE colitis with those associated with UC. The pathway enrichment analysis revealed functional similarities between inflamed regions of irAE colitis and UC. The common enriched pathways included leukocyte extravasation and immune responses, whereas non-inflamed mucosa from patients with irAE colitis was distinct from patients with UC and was characterized by the recruitment of immune cells. A similarity between the microbiota profiles was also identified. A decreased abundance of Bacteroides species was observed in inflamed regions from both irAE colitis and UC based on a microbiota composition analysis of 16S rDNA sequencing. Pathways associated with molecule transport systems, including fatty acids, were enriched in inflamed and non-inflamed irAE colitis and inflamed UC, similar to Piphillin-inferred KEGG pathways. While UC is characterized by local regions of inflammation, ICI treatment extends to non-inflammatory regions of the colonial mucosa where immune cells are reconstituted. This analysis of the similarity and heterogeneity of irAE colitis and UC provides important information for the management of irAE colitis.

Transcriptome Profiling and Metagenomic Analysis Help to Elucidate Interactions in an Inflammation-Associated Cancer Mouse Model.

Colitis is a risk factor for colorectal cancer (CRC) and can change the dynamics of gut microbiota, leading to dysbiosis and contributing to carcinogenesis. The functional interactions between colitis-associated CRC and microbiota remain unknown. In this study, colitis and CRC were induced in BALB/c mice by the administration of dextran sodium sulfate (DSS) and/or azoxymethane (AOM). Whole transcriptome profiling of normal colon was then performed, and gene set enrichment analysis (GSEA) revealed enriched fatty acid metabolism, oxidative phosphorylation, and PI3K-Akt-mTOR signaling in the tissues from DSS/AOM mice. Additionally, immunohistochemical staining showed increased expression levels of phosphorylated S6 ribosomal protein, a downstream target of the PI3K-Akt-mTOR pathway in the inflamed mucosa of DSS/AOM mice. Fecal microbes were characterized using 16S rDNA gene sequencing. Redundancy analysis demonstrated a significant dissimilarity between the DSS/AOM group and the others. Functional analysis inferred from microbial composition showed enrichments of the sphingolipid signal and lipoarabinomannan biosynthetic pathways. This study provides additional insights into alterations associated with DSS/AOM-induced colitis and associates PI3K-Akt-mTOR, sphingolipid-signaling and lipoarabinomannan biosynthetic pathways in mouse DSS/AOM-induced colitis.

Context-Specific Efficacy of Apalutamide Therapy in Preclinical Models of Pten-Deficient Prostate Cancer.

Significant improvements with apalutamide, a nonsteroidal antiandrogen used to treat patients suffering from advanced prostate cancer (PCa), have prompted evaluation for additional indications and therapeutic development with other agents; however, persistent androgen receptor (AR) signaling remains problematic. We used autochthonous mouse models of Pten-deficient PCa to examine the context-specific antitumor activity of apalutamide and profile its molecular responses. Overall, apalutamide showed potent antitumor activity in both early-stage and late-stage models of castration-naïve prostate cancer (CNPC). Molecular profiling by Western blot and immunohistochemistry associated persistent surviving cancer cells with upregulated AKT signaling. While apalutamide was ineffective in an early-stage model of castration-resistant prostate cancer (CRPC), it tended to prolong survival in late-stage CRPC. Molecular features associated with surviving cancer cells in CRPC included upregulated aberrant-AR, and phosphorylated S6 and proline-rich Akt substrate of 40 kDa (PRAS40). Strong synergy was observed with the pan-AKT inhibitor GSK690693 and apalutamide in vitro against the CNPC- and CRPC-derived cell lines and tended to improve the antitumor responses in CNPC but not CRPC in vivo. Upregulation of signal transducer and activator of transcription 3 (STAT3) and proviral insertion in murine-1 (PIM-1) were associated with combined apalutamide/GSK690693. Our findings show that apalutamide can attenuate Pten-deficient PCa in a context-specific manner and provides data that can be used to further study and, possibly, develop additional combinations with apalutamide.

Chemopreventive effects of nanoparticle curcumin in a mouse model of Pten-deficient prostate cancer.

The present study investigated the antitumor activity and chemopreventive effects of a nanoparticle formulation of curcumin in preclinical models of mouse Pten-deficient prostate cancer. The antitumor activity of the nanoparticle curcumin was evaluated in mouse castration-naïve (7113-D3) and castration-resistant prostate cancer (2945-E10) derived cell lines in vitro. Cell viability was reduced in both cell lines in a dose and time-dependent manner. The effects of long-term dietary supplementation with the nanoparticle curcumin formulation were evaluated in a conditional Pten-deficient mouse model. Prostate tissues from Pten-deficient prostate cancers were obtained after sixteen weeks of dietary supplementation of 76 mg/kg/day or 380 mg/kg/day nanoparticle curcumin. Daily supplementation of nanoparticle curcumin did not affect mouse bodyweights or spleen size but did result in enlargement of the liver. Dietary supplementation did not influence tumor burden, however, mice fed high-dose curcumin had lower cancer cell proliferation rates at 12 and 16 weeks of age. Together, these results show that daily supplementation of a nanoparticle formulation of curcumin is tolerable and suggest that curcumin could have chemopreventive activity in early-stage prostate cancer.

Targeting castration-resistant prostate cancer with androgen receptor antisense oligonucleotide therapy.

Sustained therapeutic responses from traditional and next-generation antiandrogen therapies remain elusive in clinical practice due to inherent and/or acquired resistance resulting in persistent androgen receptor (AR) activity. Antisense oligonucleotides (ASO) have the ability to block target gene expression and associated protein products and provide an alternate treatment strategy for castration-resistant prostate cancer (CRPC). We demonstrate the efficacy and therapeutic potential of this approach with a Generation-2.5 ASO targeting the mouse AR in genetically engineered models of prostate cancer. Furthermore, reciprocal feedback between AR and PI3K/AKT signaling was circumvented using a combination approach of AR-ASO therapy with the potent pan-AKT inhibitor, AZD5363. This treatment strategy effectively improved treatment responses and prolonged survival in a clinically relevant mouse model of advanced CRPC. Thus, our data provide preclinical evidence to support a combination strategy of next-generation ASOs targeting AR in combination with AKT inhibition as a potentially beneficial treatment approach for CRPC.

Generation of PTEN­knockout (­/­) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling.

Phosphatase and tensin homolog (PTEN) deficiency is associated with development, progression, and metastasis of various cancers. However, changes in gene expression associated with PTEN deficiency have not been fully characterized. To explore genes with altered expression in PTEN­deficient cells, the present study generated a PTEN­knockout cell line (ΔPTEN) from a mouse prostate cancer­derived cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR­associated protein 9 (CRISPR/Cas9) gene editing system. Following transfection of the CRISPR/Cas9 construct, DNA sequencing was performed to identify deletion of the Pten locus and PTEN inactivation was verified by western blotting. The ΔPTEN cell line exhibited enhanced RAC­alpha serine/threonine­protein kinase phosphorylation and cyclin D1 expression. In addition, an increase in cell proliferation and colony formation was observed in the ΔPTEN cell line. Gene expression profiling experiments were analyzed with microarray and microRNA (miRNA) arrays. In the microarray analysis, 111 genes exhibited ≥10­fold increased expression compared with the parent strain and mock cell line and 23 genes were downregulated. The only miRNA with increased expression of 10­fold or more was mmu­miR­210­3p. Genes with enhanced expression included genes involved in the development, progression, and metastasis of cancer such as Tet methylcytosine dioxygenase 1, twist family BHLH transcription factor 2, C­fos­induced growth factor and Wingless­Type MMTV Integration Site Family, Member 3, and genes involved in immunosuppression such as Arginase 1. The results of the present study suggest that PTEN deficiency mobilizes a variety of genes critical for cancer cell survival and host immune evasion.

Efficacy of targeted AKT inhibition in genetically engineered mouse models of PTEN-deficient prostate cancer.

The PI3K/AKT pathway is frequently altered in advanced human prostate cancer mainly through the loss of functional PTEN, and presents as potential target for personalized therapy. Our aim was to determine the therapeutic potential of the pan-AKT inhibitor, AZD5363, in PTEN-deficient prostate cancer. Here we used a genetically engineered mouse (GEM) model of PTEN-deficient prostate cancer to evaluate the in vivo pharmacodynamic and antitumor activity of AZD5363 in castration-naïve and castration-resistant prostate cancer. An additional GEM model, based on the concomitant inactivation of PTEN and Trp53 (P53), was established as an aggressive model of advanced prostate cancer and was used to further evaluate clinically relevant endpoints after treatment with AZD5363. In vivo pharmacodynamic studies demonstrated that AZD5363 effectively inhibited downstream targets of AKT. AZD5363 monotherapy significantly reduced growth of tumors in castration-naïve and castration-resistant models of PTEN-deficient prostate cancer. More importantly, AZD5363 significantly delayed tumor growth and improved overall survival and progression-free survival in PTEN/P53 double knockout mice. Our findings demonstrate that AZD5363 is effective against GEM models of PTEN-deficient prostate cancer and provide lines of evidence to support further investigation into the development of treatment strategies targeting AKT for the treatment of PTEN-deficient prostate cancer.

Evaluation of in vivo responses of sorafenib therapy in a preclinical mouse model of PTEN-deficient of prostate cancer.

BACKGROUND: Despite recent advances in the treatment for advanced prostate cancer, outcomes remain poor. This lack of efficacy has prompted the development of alternative treatment strategies. In the present study we investigate the effects of the multikinase inhibitor sorafenib in a genetically engineered mouse model of prostate cancer and explore the rational combination with the mTOR inhibitor everolimus. METHODS: Conditional prostate specific PTEN-deficient knockout mice were utilized to determine the pharmacodynamic and chemopreventive effects of sorafenib. This mouse model was also used to examine the therapeutic efficacy of sorafenib alone or in combination with everolimus. Preclinical efficacy was assessed by comparing the reduction of tumor burden, proliferation, angiogenesis and the induction of apoptosis. Molecular responses were assessed by immunohistochemical, TUNEL and western blot assays. RESULTS: Pharmacodynamic analysis revealed that a single dose of sorafenib decreased activation of the PI3K/AKT/mTOR signaling axis at doses of 30-60 mg/kg, but activated JAK/STAT3 signaling. Levels of cleaved casapase-3 increased in a dose dependent manner. Chemoprevention studies showed that chronic sorafenib administration was capable of inhibiting tumor progression through the reduction of cancer cell proliferation, angiogenesis and the induction of apoptosis. In intervention models of established castration-naïve and castration-resistant prostate cancer, treatment with sorafenib provided modest but statistically insignificant reduction in tumor burden. However, sorafenib significantly inhibited cancer cell proliferation and MVD but had minimal effects on the induction of apoptosis. Interestingly, the administration of sorafenib increased the expression levels of the androgen receptor, p-GSK3ß and p-ERK1/2 in castration-resistant prostate cancers. In both intervention models, combination therapy demonstrated a clear tendency of enhanced antitumor effects over monotherapy. Notably, the treatment combination of sorafenib and everolimus overcame therapeutic escape from single agent therapy in castration-resistant prostate cancers. CONCLUSIONS: In summary, we provide insights into the molecular responses of sorafenib therapy in a clinically relevant model of prostate cancer and present preclinical evidence for the development of targeted treatment strategies based on the use of multikinase inhibitors in combination with mTOR inhibitors for the treatment of advanced prostate cancer.

Conditional PTEN-deficient mice as a prostate cancer chemoprevention model.

BACKGROUND: We generated a mouse model of prostate cancer based on the adult-prostate-specific inactivation of phosphatase and tensin homolog (PTEN) using the Cre-loxP system. The potential of our mice as a useful animal model was examined by evaluating the chemopreventive efficacy of the anti-androgen, chlormadinone acetate (CMA). MATERIALS AND METHODS: Six-week-old mice were treated subcutaneously with 50 µg/g of CMA three times a week for 9 or 14 weeks and sacrificed at weeks 15 and 20. Macroscopic change of the entire genitourinary tract (GUT) and histologically evident prostate gland tumor development were evaluated. Proliferation and apoptosis status in the prostate were examined by immunohistochemistry. RESULTS: CMA triggered significant shrinkage of not only the GUT but also prostate glands at 15 weeks compared to the control (p=0.017 and p=0.010, respectively), and the trend became more marked after a further five-weeks of treatment. The onset of prostate adenocarcinoma was not prevented but the proliferation of cancer cells was inhibited by CMA, which suggested the androgen axis is critical for cancer growth in these mice. CONCLUSIONS: Conditional PTEN-deficient mice are useful as a preclinical model for chemoprevention studies and serve as a valuable tool for the future screening of potential chemopreventive agents.