Defining the challenges and opportunities for using patient-derived models in prostate cancer research.

BACKGROUND: There are relatively few widely used models of prostate cancer compared to other common malignancies. This impedes translational prostate cancer research because the range of models does not reflect the diversity of disease seen in clinical practice. In response to this challenge, research laboratories around the world have been developing new patient-derived models of prostate cancer, including xenografts, organoids, and tumor explants. METHODS: In May 2023, we held a workshop at the Monash University Prato Campus for researchers with expertise in establishing and using a variety of patient-derived models of prostate cancer. This review summarizes our collective ideas on how patient-derived models are currently being used, the common challenges, and future opportunities for maximizing their usefulness in prostate cancer research. RESULTS: An increasing number of patient-derived models for prostate cancer are being developed. Despite their individual limitations and varying success rates, these models are valuable resources for exploring new concepts in prostate cancer biology and for preclinical testing of potential treatments. Here we focus on the need for larger collections of models that represent the changing treatment landscape of prostate cancer, robust readouts for preclinical testing, improved in vitro culture conditions, and integration of the tumor microenvironment. Additional priorities include ensuring model reproducibility, standardization, and replication, and streamlining the exchange of models and data sets among research groups. CONCLUSIONS: There are several opportunities to maximize the impact of patient-derived models on prostate cancer research. We must develop large, diverse and accessible cohorts of models and more sophisticated methods for emulating the intricacy of patient tumors. In this way, we can use the samples that are generously donated by patients to advance the outcomes of patients in the future.

Peroxisomal ß-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer.

BACKGROUND: Peroxisomes are central metabolic organelles that have key roles in fatty acid homoeostasis. As prostate cancer (PCa) is particularly reliant on fatty acid metabolism, we explored the contribution of peroxisomal ß-oxidation (perFAO) to PCa viability and therapy response. METHODS: Bioinformatic analysis was performed on clinical transcriptomic datasets to identify the perFAO enzyme, 2,4-dienoyl CoA reductase 2 (DECR2) as a target gene of interest. Impact of DECR2 and perFAO inhibition via thioridazine was examined in vitro, in vivo, and in clinical prostate tumours cultured ex vivo. Transcriptomic and lipidomic profiling was used to determine the functional consequences of DECR2 inhibition in PCa. RESULTS: DECR2 is upregulated in clinical PCa, most notably in metastatic castrate-resistant PCa (CRPC). Depletion of DECR2 significantly suppressed proliferation, migration, and 3D growth of a range of CRPC and therapy-resistant PCa cell lines, and inhibited LNCaP tumour growth and proliferation in vivo. DECR2 influences cell cycle progression and lipid metabolism to support tumour cell proliferation. Further, co-targeting of perFAO and standard-of-care androgen receptor inhibition enhanced suppression of PCa cell proliferation. CONCLUSION: Our findings support a focus on perFAO, specifically DECR2, as a promising therapeutic target for CRPC and as a novel strategy to overcome lethal treatment resistance.

Fatty acid elongation regulates mitochondrial ß-oxidation and cell viability in prostate cancer by controlling malonyl-CoA levels.

Recently, the fatty acid elongation enzyme ELOVL5 was identified as a critical pro-metastatic factor in prostate cancer, required for cell growth and mitochondrial homeostasis. The fatty acid elongation reaction catalyzed by ELOVL5 utilizes malonyl-CoA as the carbon donor. Here, we demonstrate that ELOVL5 knockdown causes malonyl-CoA accumulation. Malonyl-CoA is a cellular substrate that can inhibit fatty acid ß-oxidation in the mitochondria through allosteric inhibition of carnitine palmitoyltransferase 1A (CPT1A), the enzyme that controls the rate-limiting step of the long chain fatty acid ß-oxidation cycle. We hypothesized that changes in malonyl-CoA abundance following ELOVL5 knockdown could influence mitochondrial ß-oxidation rates in prostate cancer cells, and regulate cell viability. Accordingly, we find that ELOVL5 knockdown is associated with decreased mitochondrial ß-oxidation in prostate cancer cells. Combining ELOVL5 knockdown with FASN inhibition to increase malonyl-CoA abundance endogenously enhances the effect of ELOVL5 knockdown on prostate cancer cell viability, while preventing malonyl-CoA production rescues the cells from the effect of ELOVL5 knockdown. Our findings indicate an additional role for fatty acid elongation, in the control of malonyl-CoA homeostasis, alongside its established role in the production of long-chain fatty acid species, to explain the importance of fatty acid elongation for cell viability.

Spatial MS multiomics on clinical prostate cancer tissues.

Mass spectrometry (MS) and MS imaging (MSI) are used extensively for both the spatial and bulk characterization of samples in lipidomics and proteomics workflows. These datasets are typically generated independently due to different requirements for sample preparation. However, modern omics technologies now provide higher sample throughput and deeper molecular coverage, which, in combination with more sophisticated bioinformatic and statistical pipelines, make generating multiomics data from a single sample a reality. In this workflow, we use spatial lipidomics data generated by matrix-assisted laser desorption/ionization MSI (MALDI-MSI) on prostate cancer (PCa) radical prostatectomy cores to guide the definition of tumor and benign tissue regions for laser capture microdissection (LCM) and bottom-up proteomics all on the same sample and using the same mass spectrometer. Accurate region of interest (ROI) mapping was facilitated by the SCiLS region mapper software and dissected regions were analyzed using a dia-PASEF workflow. A total of 5525 unique protein groups were identified from all dissected regions. Lysophosphatidylcholine acyltransferase 1 (LPCAT1), a lipid remodelling enzyme, was significantly enriched in the dissected regions of cancerous epithelium (CE) compared to benign epithelium (BE). The increased abundance of this protein was reflected in the lipidomics data with an increased ion intensity ratio for pairs of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) in CE compared to BE.

Targeting hyaluronan-mediated motility receptor (HMMR) enhances response to androgen receptor signalling inhibitors in prostate cancer.

BACKGROUND: Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs. METHODS: RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs). RESULTS: HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs. CONCLUSION: Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs.

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

Relationship Power, Antiretroviral Adherence, and Physical and Mental Health Among Women Living with HIV in Rural Kenya.

Little is known about the association of gender-based power imbalances and health and health behaviors among women with HIV (WWH). We examined cross-sectional baseline data among WWH in a cluster-randomized control trial (NCT02815579) in rural Kenya. We assessed associations between the Sexual Relationship Power Scale (SRPS) and ART adherence, physical and mental health, adjusting for sociodemographic and social factors. SRPS consists of two subscales: relationship control (RC) and decision-making dominance. Women in the highest and middle tertiles for RC had a 7.49 point and 8.88 point greater Medical Outcomes Study-HIV mental health score, and a 0.27 and 0.29 lower odds of depression, respectively, compared to women in the lowest tertile. We did not find associations between SPRS or its subscales and ART adherence. Low sexual relationship power, specifically low RC, may be associated with poor mental health among WWH. Intervention studies aimed to improve RC among WWH should be studied to determine their effect on improving mental health.

RESUMEN: Poco se sabe acerca de su asociación con los desequilibrios de poder basados en el género y los comportamientos de salud y salud entre las mujeres con Virus de Inmunodeficiencia Humana (VIH). Examinamos los datos de referencia transversales entre mujeres con VIH en un ensayo de control aleatorizado por grupos (NCT02815579) en las zonas rurales de Kenia. Evaluamos las asociaciones entre la Escala de Poder de Relación Sexual y la adherencia a la Terapia Antirretroviral (TAR), la salud física y mental, ajustando por factores sociodemográficos y sociales. La Escala de Poder de Relación Sexual consiste de dos subescalas: control de relaciones y dominio en la toma de decisiones. Las mujeres en los terciles más alto y mediano para control de relaciones tenían una puntuación de salud mental de 7.49 puntos y 8.88 puntos mayor en el Medical Outcomes Study HIV Health Survey (MOS)-HIV, y una puntuación de salud mental de 0.27 y 0.29 menores probabilidades de depresión, respectivamente, en comparación con las mujeres en el tercil más bajo. El bajo poder de relación sexual, específicamente el control de relaciones bajo, puede estar asociado con una salud mental deficiente entre las mujeres con VIH. Se deben estudiar estudios de intervención destinados a mejorar el control de relaciones entre mujeres con VIH para determinar su efecto en mejorar la salud mental.

Incidence of HIV disclosure among HIV affected heterosexual partners using a community health worker led mechanism in rural Uganda; a quasi-experimental study.

BACKGROUND: HIV disclosure is vital in HIV management. Community Health Workers (CHW) were reported to support partner disclosure among HIV affected heterosexual partners with disclosure difficulties. However, time to disclosure attributed to use of CHW led disclosure support mechanism was not documented. This study compared the incidence of sexual partner disclosure among adults living with HIV (ALHIV) with CHW support and those without in the greater Luwero region, Uganda. METHODS: We conducted a quasi-experimental study with two arms allocated by geographically determined clusters and adjusted for between-group differences; among ALHIV in the greater Luwero region of Uganda who had never disclosed to their current primary sexual partners. We allocated study clusters to either a CHW-led intervention or control arm. In both arms, we consecutively recruited participants; those in the intervention arm received CHW disclosure support in addition to routine care. The overall follow-up was six months, and the primary outcome was disclosure to the partner. We used survival analysis with proportional hazard ratios to determine the time to partner disclosure in both arms. RESULTS: A total of 245 participants were enrolled, and 230 (93.9%) completed the study; of these, 112 (48.7%) were in the intervention and 118 (51.3%) in the control arm. The mean age was 31 ± 8 years with a range of 18 to 55 years; the majority were females, 176 (76.5%). The cumulative incidence of disclosure was higher in the intervention arm, 8.76 [95% CI: 7.20-10.67] per 1,000 person-days versus 5.15 [95%CI: 4.85-6.48] per 1,000 person-days in the control arm, log-rank test, X2 = 12.93, P < 0.001. Male gender, aHR = 1.82, tertiary education, aHR = 1.51, and relationship duration of > six months, aHR = 1.19 predicted disclosure. Prior disclosure to a relative, aHR = 0.55, and having more than one sexual partner in the past three months, aHR = 0.74, predicted non-disclosure. CONCLUSION: CHW-led support mechanism increased the rate of sexual partner disclosure among ALHIV with disclosure difficulties. Therefore, to achieve the global targets of ending HIV, near location CHW-led disclosure support mechanism may be used to hasten HIV disclosure in rural settings.

Experiences and challenges of using community health worker-led mechanism in supporting HIV disclosure among adults living with HIV in heterosexual relationships in the rural Uganda.

BACKGROUND: HIV status disclosure among sexual partners is vital in HIV management. Community health workers (CHW) support HIV disclosure among adults living with HIV (ALHIV) in sexual relationships with disclosure difficulties. However, experiences and challenges of using CHW-led disclosure support mechanism were not documented. This study explored experiences and challenges involved in using CHW-led disclosure support mechanism among ALHIV in heterosexual relationships in the rural Uganda. METHODS: This was a phenomenological qualitative study involving in-depth interviews among CHWs and ALHIV with HIV disclosure difficulties to sexual partners in greater Luwero region, Uganda. We conducted 27 interviews among purposively selected CHWs and participants who had participated in the CHW-led disclosure support mechanism. Interviews were conducted until saturation was reached; and analysis was done using inductive and deductive content analysis in Atlas. RESULTS: All respondents viewed HIV disclosure as an important strategy in HIV management. Provision of adequate counseling and support to those intending to disclose was instrumental for successful disclosure. However, fear of the negative disclosure outcomes was viewed as a barrier to disclosure. The CHWs were viewed as having an added advantage in supporting disclosure as opposed to the routine disclosure counseling. However, HIV disclosure using CHW-led support mechanism would be limited by possible bleach of client's confidentiality. Therefore, respondents thought that appropriate selection of CHWs would improve their trust in the community. Additionally, providing CHWs with adequate training and facilitation during the disclosure support mechanism was viewed to improve their work. CONCLUSION: Community health workers were viewed as being more supportive in HIV disclosure among ALHIV with disclosure difficulties to sexual partners compared to routine facility based disclosure counseling. Therefore, near location CHW-led disclosure mechanism was acceptable and useful in supporting HIV disclosure among HIV-affected sexual partners in rural settings.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor ß and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Combined impact of lipidomic and genetic aberrations on clinical outcomes in metastatic castration-resistant prostate cancer.

BACKGROUND: Both changes in circulating lipids represented by a validated poor prognostic 3-lipid signature (3LS) and somatic tumour genetic aberrations are individually associated with worse clinical outcomes in men with metastatic castration-resistant prostate cancer (mCRPC). A key question is how the lipid environment and the cancer genome are interrelated in order to exploit this therapeutically. We assessed the association between the poor prognostic 3-lipid signature (3LS), somatic genetic aberrations and clinical outcomes in mCRPC. METHODS: We performed plasma lipidomic analysis and cell-free DNA (cfDNA) sequencing on 106 men with mCRPC commencing docetaxel, cabazitaxel, abiraterone or enzalutamide (discovery cohort) and 94 men with mCRPC commencing docetaxel (validation cohort). Differences in lipid levels between men ± somatic genetic aberrations were assessed with t-tests. Associations between the 3LS and genetic aberrations with overall survival (OS) were examined using Kaplan-Meier methods and Cox proportional hazard models. RESULTS: The 3LS was associated with shorter OS in the discovery (hazard ratio [HR] 2.15, 95% confidence interval [CI] 1.4-3.3, p < 0.001) and validation cohorts (HR 2.32, 95% CI 1.59-3.38, p < 0.001). Elevated plasma sphingolipids were associated with AR, TP53, RB1 and PI3K aberrations (p < 0.05). Men with both the 3LS and aberrations in AR, TP53, RB1 or PI3K had shorter OS than men with neither in both cohorts (p ≤ 0.001). The presence of 3LS and/or genetic aberration was independently associated with shorter OS for men with AR, TP53, RB1 and PI3K aberrations (p < 0.02). Furthermore, aggressive-variant prostate cancer (AVPC), defined as 2 or more aberrations in TP53, RB1 and/or PTEN, was associated with elevated sphingolipids. The combination of AVPC and 3LS predicted for a median survival of ~12 months. The relatively small sample size of the cohorts limits clinical applicability and warrants future studies. CONCLUSIONS: Elevated circulating sphingolipids were associated with AR, TP53, RB1, PI3K and AVPC aberrations in mCRPC, and the combination of lipid and genetic abnormalities conferred a worse prognosis. These findings suggest that certain genotypes in mCRPC may benefit from metabolic therapies.

Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone.

The latency associated with bone metastasis emergence in castrate-resistant prostate cancer is attributed to dormancy, a state in which cancer cells persist prior to overt lesion formation. Using single-cell transcriptomics and ex vivo profiling, we have uncovered the critical role of tumor-intrinsic immune signaling in the retention of cancer cell dormancy. We demonstrate that loss of tumor-intrinsic type I IFN occurs in proliferating prostate cancer cells in bone. This loss suppresses tumor immunogenicity and therapeutic response and promotes bone cell activation to drive cancer progression. Restoration of tumor-intrinsic IFN signaling by HDAC inhibition increased tumor cell visibility, promoted long-term antitumor immunity, and blocked cancer growth in bone. Key findings were validated in patients, including loss of tumor-intrinsic IFN signaling and immunogenicity in bone metastases compared to primary tumors. Data herein provide a rationale as to why current immunotherapeutics fail in bone-metastatic prostate cancer, and provide a new therapeutic strategy to overcome the inefficacy of immune-based therapies in solid cancers.

Water Insecurity is Associated with Lack of Viral Suppression and Greater Odds of AIDS-Defining Illnesses Among Adults with HIV in Western Kenya.

Reliable access to safe and acceptable water in sufficient quantities (i.e., water security) is important for medication adherence and limiting pathogen exposure, yet prior studies have only considered the role of food security as a social determinant of HIV-related health. Therefore, the objective of this analysis was to assess the relationships between household water insecurity and HIV-related outcomes among adults living with HIV in western Kenya (N = 716). We conducted a cross-sectional analysis of baseline data from Shamba Maisha (NCT02815579), a cluster randomized controlled trial of a multisectoral agricultural and asset loan intervention. Baseline data were collected from June 2016 to December 2017. We assessed associations between water insecurity and HIV-related outcomes, adjusting for clinical and behavioral confounders, including food insecurity. Each five-unit higher household water insecurity score (range: 0-51) was associated with 1.21 higher odds of having a viral load ≥ 1000 copies/mL (95% CI 1.07, 1.36) and 1.26 higher odds of AIDS-defining illness (95% CI 1.11, 1.42). Household water insecurity was not associated with CD4 cell count (B: 0.27; 95% CI -3.59, 13.05). HIV treatment and support programs should consider assessing and addressing water insecurity in addition to food insecurity to optimize HIV outcomes.

RESUMEN: El acceso seguro al agua potable en cantidades suficientes (es decir, seguridad hídrica) es importante para la adherencia a la medicación y para limitar la exposición a patógenos; sin embargo, estudios anteriores solo han considerado el papel de la seguridad alimentaria como un determinante social de salud relacionado con el VIH. Por lo tanto, el objetivo de este análisis fue evaluar las relaciones entre la inseguridad hídrica en hogares y los resultados relacionados con el VIH en adultos que viven con VIH en el oeste de Kenia (N = 716). Realizamos un análisis transversal de los datos basales de la iniciativa Shamba Maisha (NCT02815579), un ensayo controlado aleatorio por conglomerados de una intervención multisectorial de créditos para insumos agrícolas. Los datos basales se recopilaron de junio de 2016 a diciembre de 2017. Evaluamos las asociaciones entre la inseguridad hídrica y resultados relacionados con el VIH, ajustando por factores de confusión clínicos y conductuales, incluyendo inseguridad alimentaria. Cada cinco unidades superiores de puntajes de inseguridad hídrica doméstica (rango: 0-51) fue asociado con 1.21 mayores probabilidades de tener una carga viral más alta ≥ 1000 copias / ml (CI 95%: 1,07-1,36) y con 1.26 mayores probabilidades de factores determinantes del SIDA (CI 95%: 1,11-1,42). La inseguridad de hídrica doméstica no se asoció con el recuento de células CD4 (B: −0,27; CI 95%: -13,59-13,05). Los programas de tratamiento y de apoyo al VIH deben considerar evaluar y abordar la inseguridad hídrica además de la inseguridad alimentaria para optimizar los resultados del VIH.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

Inhibition of de novo lipogenesis targets androgen receptor signaling in castration-resistant prostate cancer.

A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119-mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7-driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.

Removal of optimal cutting temperature (O.C.T.) compound from embedded tissue for MALDI imaging of lipids.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a common molecular imaging modality used to characterise the abundance and spatial distribution of lipids in situ. There are several technical challenges predominantly involving sample pre-treatment and preparation which have complicated the analysis of clinical tissues by MALDI-MSI. Firstly, the common embedding of samples in optimal cutting temperature (O.C.T.), which contains high concentrations of polyethylene glycol (PEG) polymers, causes analyte signal suppression during mass spectrometry (MS) by competing for available ions during ionisation. This suppressive effect has constrained the application of MALDI-MSI for the molecular mapping of clinical tissues. Secondly, the complexity of the mass spectra is obtained by the formation of multiple adduct ions. The process of analyte ion formation during MALDI can generate multiple m/z peaks from a single lipid species due to the presence of alkali salts in tissues, resulting in the suppression of protonated adduct formation and the generation of multiple near isobaric ions which produce overlapping spatial distributions. Presented is a method to simultaneously remove O.C.T. and endogenous salts. This approach was applied to lipid imaging in order to prevent analyte suppression, simplify data interpretation, and improve sensitivity by promoting lipid protonation and reducing the formation of alkali adducts.

eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA.

Emerging advances in cancer therapy have transformed the landscape towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are 'game-changers' for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that negatively modulates the elongation stage of protein synthesis, promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5'-UTR (5'-untranslated region) which starts with a non-canonical CUG as the initiation codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5'-region. This, and other roles of eEF2K in cancer cell biology (e.g. in cell survival and migration), may be exploited for the design of future therapeutic strategies.

Elevated levels of tumour apolipoprotein D independently predict poor outcome in breast cancer patients.

AIMS: Apolipoprotein D (ApoD) is a protein that is regulated by androgen and oestrogen, and is a major constituent of breast cysts. Although ApoD has been reported to be a marker of breast cancer, its prognostic importance in invasive breast cancer is unclear. The aim of this study was to investigate the relationship between ApoD protein expression, oestrogen receptor-α (ERα) expression and androgen receptor (AR) expression in predicting breast cancer outcome. METHODS AND RESULTS: ApoD levels were measured by the use of immunohistochemistry and video image analysis on tissue sections from a breast cancer cohort (n = 214). We assessed the associations of ApoD expression with disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS). We also assessed the relationship between ApoD expression, AR expression and ERα expression in predicting OS. ApoD expression (>1% ApoD positivity) was found in 72% (154/214) of tissues. High ApoD positivity (≥20.7%, fourth quartile) was an independent predictor of MFS and OS, and conferred a 2.2-fold increased risk of developing metastatic disease and a 2.1-fold increased risk of breast cancer-related death. ApoD positivity was not associated with AR or ERα nuclear positivity. However, patients with (≥1%) ERα-positive cancers with low (<20.7%) ApoD positivity, or those showing high (≥78%) AR positivity and low (<20.7%) ApoD positivity had better OS than other patient groups. CONCLUSIONS: ApoD expression could be used to predict breast cancer prognosis independently of ERα and AR expression.

Identification of Novel Response and Predictive Biomarkers to Hsp90 Inhibitors Through Proteomic Profiling of Patient-derived Prostate Tumor Explants.

Inhibition of the heat shock protein 90 (Hsp90) chaperone is a promising therapeutic strategy to target expression of the androgen receptor (AR) and other oncogenic drivers in prostate cancer cells. However, identification of clinically-relevant responses and predictive biomarkers is essential to maximize efficacy and treatment personalization. Here, we combined mass spectrometry (MS)-based proteomic analyses with a unique patient-derived explant (PDE) model that retains the complex microenvironment of primary prostate tumors. Independent discovery and validation cohorts of PDEs (n = 16 and 30, respectively) were cultured in the absence or presence of Hsp90 inhibitors AUY922 or 17-AAG. PDEs were analyzed by LC-MS/MS with a hyper-reaction monitoring data independent acquisition (HRM-DIA) workflow, and differentially expressed proteins identified using repeated measure analysis of variance (ANOVA; raw p value <0.01). Using gene set enrichment, we found striking conservation of the most significantly AUY922-altered gene pathways between the discovery and validation cohorts, indicating that our experimental and analysis workflows were robust. Eight proteins were selectively altered across both cohorts by the most potent inhibitor, AUY922, including TIMP1, SERPINA3 and CYP51A (adjusted p < 0.01). The AUY922-mediated decrease in secretory TIMP1 was validated by ELISA of the PDE culture medium. We next exploited the heterogeneous response of PDEs to 17-AAG in order to detect predictive biomarkers of response and identified PCBP3 as a marker with increased expression in PDEs that had no response or increased in proliferation. Also, 17-AAG treatment led to increased expression of DNAJA1 in PDEs that exhibited a cytostatic response, revealing potential drug resistance mechanisms. This selective regulation of DNAJA1 was validated by Western blot analysis. Our study establishes "proof-of-principle" that proteomic profiling of drug-treated PDEs represents an effective and clinically-relevant strategy for identification of biomarkers that associate with certain tumor-specific responses.

An analysis of a multiple biomarker panel to better predict prostate cancer metastasis after radical prostatectomy.

A plethora of individual candidate biomarkers for predicting biochemical relapse in localized prostate cancer (PCa) have been proposed. Combined biomarkers may improve prognostication, and ensuring validation against more clinically relevant endpoints are required. The Australian PCa Research Centre NSW has contributed to numerous studies of molecular biomarkers associated with biochemical relapse. In the current study, these biomarkers were re-analyzed for biochemical relapse, metastatic relapse and PCa death with extended follow-up. Biomarkers of significance were then used to develop a combined prognostic model for clinical outcomes and validated in a large independent cohort. The discovery cohort (n = 324) was based on 12 biomarkers with a median follow-up of 16 years. Seven biomarkers were significantly associated with biochemical relapse. Three biomarkers were associated with metastases: AZGP1, Ki67 and PML. Only AZGP1 was associated with PCa death. In their individual and combinational forms, AZGP1 and Ki67 as a dual BM signature was the most robust predictor of metastatic relapse (AUC 0.762). The AZPG1 and Ki67 signature was validated in an independent cohort of 347 PCa patients. The dual BM signature of AZGP1 and Ki67 predicted metastasis in the univariable (HR 7.2, 95% CI, 1.6-32; p = 0.01) and multivariable analysis (HR 5.4, 95% CI, 1.2-25; p = 0.03). The dual biomarker signature marginally improved risk prediction compared to AZGP1 alone (AUC 0.758 versus 0.738, p < 0.001). Our findings indicate that biochemical relapse is not an adequate surrogate for metastasis or PCa death. The dual biomarker signature of AZGP1 and Ki67 offers a small benefit in predicting metastasis over AZGP1 alone.