Aneustat (OMN54) has aerobic glycolysis-inhibitory activity and also immunomodulatory activity as indicated by a first-generation PDX prostate cancer model.

Aneustat (OMN54) is a multivalent, botanical anticancer candidate therapeutic. A recent Phase-I clinical trial has indicated that it is well tolerated by patients and has immunomodulatory activity. In our study, using in vitro and in vivo prostate cancer models, we investigated Aneustat with regard to effects on (i) cancer-generated immunosuppression based on aerobic glycolysis leading to acidification of the tumor microenvironment, and (ii) immune-related processes such as macrophage differentiation and shifts in the intratumoral levels of host immune cells. Aneustat markedly reduced glucose consumption, lactic acid secretion, glycolysis-related gene expressions and proliferation of human LNCaP prostate cancer cells. In addition, Aneustat induced differentiation of RAW264.7 macrophages to the M1 anticancer phenotype. Treatment of LNCaP xenografts and first-generation patient-derived prostate cancer tissue xenografts with Aneustat in both cases led to a marked shift in intratumoral host (mouse/patient) immune cell levels: a higher ratio of cytotoxic CD8+ T/Treg cells, higher numbers of NK cells, lower numbers of Treg cells and MDSCs, i.e. changes favoring the host anticancer immune response. Taken together, the data indicate that Aneustat has immunomodulatory activity based on inhibition of aerobic glycolysis which in patients may lead to reduction of cancer-induced immunosuppression. Furthermore, first-generation patient-derived cancer tissue xenograft models may be used for screening compounds for immunomodulatory activity.

Treatment with docetaxel in combination with Aneustat leads to potent inhibition of metastasis in a patient-derived xenograft model of advanced prostate cancer.

BACKGROUND: Docetaxel used for first-line treatment of advanced prostate cancer (PCa) is only marginally effective. We previously showed, using the LTL-313H subrenal capsule patient-derived metastatic PCa xenograft model, that docetaxel combined with Aneustat (OMN54), a multivalent plant-derived therapeutic, led to marked synergistic tumour growth inhibition. Here, we investigated the effect of docetaxel+Aneustat on metastasis. METHODS: C4-2 cells were incubated with docetaxel, Aneustat and docetaxel+Aneustat to assess effects on cell migration. The LTL-313H model, similarly treated, was analysed for effects on lung micro-metastasis and kidney invasion. The LTL-313H gene expression profile was compared with profiles of PCa patients (obtained from Oncomine) and subjected to IPA to determine involvement of cancer driver genes. RESULTS: Docetaxel+Aneustat markedly inhibited C4-2 cell migration and LTL-313H lung micro-metastasis/kidney invasion. Oncomine analysis indicated that treatment with docetaxel+Aneustat was associated with improved patient outcome. The drug combination markedly downregulated expression of cancer driver genes such as FOXM1 (and FOXM1-target genes). FOXM1 overexpression reduced the anti-metastatic activity of docetaxel+Aneustat. CONCLUSIONS: Docetaxel+Aneustat can inhibit PCa tissue invasion and metastasis. This activity appears to be based on reduced expression of cancer driver genes such as FOXM1. Use of docetaxel+Aneustat may provide a new, more effective regimen for therapy of metastatic PCa.

Subrenal capsule grafting technology in human cancer modeling and translational cancer research.

Patient-derived xenograft (PDX) cancer models with high fidelity are in great demand. While the majority of PDXs are grafted under the skin of immunodeficient mice, the Living Tumor Laboratory (LTL), using unique subrenal capsule grafting techniques, has successfully established more than 200 transplantable PDX models of various low to high grade human cancers. The LTL PDX models retain key biological properties of the original malignancies, including histopathological and molecular characteristics, tumor heterogeneity, metastatic ability, and response to treatment. The PDXs are stored frozen at early transplant generations in a resurrectable form, which eliminates continuous passaging in mice, thus ensuring maintenance of the high biologic and molecular fidelity and reproducibility of the models. The PDX models have been demonstrated to be powerful tools for (i) studies of cancer progression, metastasis and drug resistance, (ii) evidenced-based precision cancer therapy, (iii) preclinical drug efficacy testing and discovery of new anti-cancer drug candidates. To better provide resources for the research community, an LTL website (www.livingtumorlab.com) has been designed as a publicly accessible database which allows researchers to identify PDX models suitable for translational/preclinical cancer research. In summary, subrenal capsule grafting technology maximizes both tumor engraftment rate and retention of human cancer heterogeneity. Moreover, the method makes possible the recovery of PDXs from frozen stocks for further applications, thus providing a powerful platform for translational cancer research.

Elevated expression of the centromere protein-A(CENP-A)-encoding gene as a prognostic and predictive biomarker in human cancers.

Centromere protein-A (CENP-A), a histone-H3 variant, plays an essential role in cell division by ensuring proper formation and function of centromeres and kinetochores. Elevated CENP-A expression has been associated with cancer development. This study aimed to establish whether elevated CENP-A expression can be used as a prognostic and predictive cancer biomarker. Molecular profiling of CENP-A in human cancers was investigated using genomic, transcriptomic and patient information from databases, including COSMIC, Oncomine, Kaplan-Meier plotter and cBioPortal. A network of CENP-A co-expressed genes was derived from cBioPortal and analyzed using Ingenuity Pathway Analysis (IPA) and Oncomine protocols to explore the function of CENP-A and its predictive potential. Transcriptional and post-transcriptional regulation of CENP-A expression was analyzed in silico. It was found that CENP-A expression was elevated in 20 types of solid cancer compared with normal counterparts. Elevated CENP-A expression highly correlated with cancer progression and poor patient outcome. Genomic analysis indicated that the elevated CENP-A expression was not due to alterations in the sequence or copy number of the CENP-A gene. Furthermore, CENP-A can be regulated by key oncogenic proteins and tumor-suppressive microRNAs. CENP-A co-expression network analysis indicated that CENP-A function is associated with cell cycle progression. Oncomine analysis showed a strong correlation between elevated CENP-A expression and oncolytic response of breast cancer patients to taxane-based chemotherapy. In conclusion, elevated CENP-A expression is coupled to malignant progression of numerous types of cancer. It may be useful as a biomarker of poor patient prognosis and as a predictive biomarker for taxane-based chemotherapy.

Cancer-generated lactic acid: a regulatory, immunosuppressive metabolite?

The common preference of cancers for lactic acid-generating metabolic energy pathways has led to proposals that their reprogrammed metabolism confers growth advantages such as decreased susceptibility to hypoxic stress. Recent observations, however, suggest that it generates a novel way for cancer survival. There is increasing evidence that cancers can escape immune destruction by suppressing the anti-cancer immune response through maintaining a relatively low pH in their micro-environment. Tumours achieve this by regulating lactic acid secretion via modification of glucose/glutamine metabolisms. We propose that the maintenance by cancers of a relatively low pH in their micro-environment, via regulation of their lactic acid secretion through selective modification of their energy metabolism, is another major mechanism by which cancers can suppress the anti-cancer immune response. Cancer-generated lactic acid could thus be viewed as a critical, immunosuppressive metabolite in the tumour micro-environment rather than a 'waste product'. This paradigm shift can have major impact on therapeutic strategy development.

Lessons from in-vivo models of castration-resistant prostate cancer.

PURPOSE OF REVIEW: Although the treatment of castration-resistant prostate cancer (CRPC) has benefited from the use of increasingly potent androgen synthesis inhibitors and androgen receptor (AR) antagonists, it is only marginally effective. There is therefore a critical need for a better understanding of the mechanisms underlying the CRPC development and more effective therapeutic approaches. Here, we focus on the advancements reported in the last 18 months, particularly with regard to the mechanisms of castration resistance and potential therapeutic targets emerging from the studies with in-vivo models. RECENT FINDINGS: Recent findings indicate that AR-dependent mechanisms, for example, increased expression of CYP17A1 and AR splice variants, play important roles in in-vivo castration resistance to new antiandrogens and androgen synthesis inhibitors. Whereas current therapeutic approaches focus on AR-dependent CRPC, studies based on genetically engineered mouse models indicate that castration resistance can develop in the absence of robust AR signaling. Furthermore, increasing evidence suggests that cellular plasticity of prostate adenocarcinoma allows AR-independent CRPC development via various adaptive mechanisms. SUMMARY: Significant progress has been made in the understanding of AR-dependent and AR-independent mechanisms involved in the development of CRPC. This may lead to identification of new therapeutic targets and improved therapy.

Epithelial immune cell-like transition (EIT): a proposed transdifferentiation process underlying immune-suppressive activity of epithelial cancers.

The immune system plays a key role in eliminating cancer cells in the body. However, even in fully immune-competent bodies cancers can evade anti-tumor immune action. There is increasing evidence that epithelial cancers can actively suppress anti-tumor immune responses by creating an immune-suppressive micro-environment. It has been reported that epithelial cancers can express immune genes/proteins not normally expressed by their parental tissues, including a variety of cytokines/receptors, immune transcription factors and Ig motifs in cell surface molecules. Recently we observed increased expression of immune genes, including immune-suppressive genes, by prostate epithelial cancers. In view of the above, we propose that immune-suppressive activity of epithelial cancers may stem from their acquisition of immune properties via a transdifferentiation process, we term "Epithelial Immune Cell-like Transition" (EIT), similar to neuroendocrine-like transdifferentiation of prostate adenocarcinoma cells. We propose that the acquired immune properties enable the cancer cells to "communicate" with immune cells, leading to suppression of anti-cancer immune activity in their micro-environment and facilitation of the expansion and malignant progression of the disease. Acquired immune properties of epithelial cancers, which might be quite common, could provide novel targets for reducing cancer-generated immune-suppressive activity and enhancing anti-tumor immune activity. This proposed paradigm shift could lead to novel therapeutic approaches with improved efficacy and broad application.

Use of irinotecan for treatment of small cell carcinoma of the prostate.

BACKGROUND: Prostatic small cell carcinoma (SCC) is a rare variant of prostate cancer. It is extremely aggressive and resistant to available therapies with a median survival range of 5-17 months. No standard chemotherapeutic regimen has been established for its treatment. In search of a new therapeutic approach, we examined the response of patient-derived prostatic SCC tissue xenografts to irinotecan, a topoisomerase I inhibitor. METHODS: A tumor tissue line was established from a patient's prostatic SCC by subrenal capsule grafting using NOD-SCID mice. Mice carrying subcutaneous transplants of the tumor line were then treated for 2 weeks with irinotecan alone and in combination with cisplatin. The effect on tumor volume, histopathology, and apoptosis were determined. RESULTS: The prostatic SCC tissue line resembled the donor tissue in morphologic and immunohistochemical features. Irinotecan (20 mg/kg/day; days 1-3, 8-10) completely arrested xenograft growth with a small reduction in tumor volume and only minor weight loss of the hosts (7%); irinotecan (12 mg/kg; same schedule) + cisplatin (2.5 mg/kg/day; days 1 and 8) had a similar effect, but with lower weight loss. While the growth inhibition involved apoptosis, it was also associated with a marked increase in autophagy. CONCLUSIONS: Tumor tissue lines established via subrenal capsule xenografting provide models with clinical relevance and the present study suggests that irinotecan could be useful for therapy of refractory prostatic SCC, in particular in combination with cisplatin.

Development of metastatic and non-metastatic tumor lines from a patient's prostate cancer specimen-identification of a small subpopulation with metastatic potential in the primary tumor.

BACKGROUND: Metastasis is the major cause of prostate cancer deaths. Tumor heterogeneity in both primary and metastatic prostate cancers is a major hurdle in elucidating the mechanisms of metastasis in this disease. To circumvent this obstacle and improve our understanding of prostate cancer metastasis, we developed multiple tumor tissue lines from one patient's primary prostate cancer specimen to examine differences in metastatic ability. METHODS: Pieces of tissue from different foci of a patient's primary prostate tumor were grafted into subrenal capsules of NOD-SCID mice and transplantable tumor sublines were established by serial passage. The metastatic ability of each subline was tested via orthotopic grafting into mice. Chromosomal alterations exclusively presented in a metastatic subline were examined by SKY and investigated for their presence in parental tissues by fluorescence in situ hybridization. RESULTS: Three transplantable sublines were developed, resembling the primary tumor histologically, exhibiting poor differentiation, and different growth rates. Importantly, the LTL-220N and LTL-221N sublines were non-metastatic, whereas the LTL-220M subline was spontaneously metastatic in vivo. SKY analysis showed limited but unique chromosomal alterations in each subline. Some chromosomal alterations, exclusively present in the metastatic LTL-220M subline, were also observed in a small portion of the parental cancer tissues. CONCLUSION: The results indicate that in primary prostate tumors metastatic potential can be confined to a minority of cancer cells. Subrenal capsule xenograft methodology can be used to dissect heterogeneous cancer cells in a patient's primary tumor and sublines derived from such cells provide valuable tools for investigating mechanisms underlying prostate cancer metastasis.

Pharmacogenomic approach reveals a role for the x(c)- cystine/glutamate antiporter in growth and celastrol resistance of glioma cell lines.

The x(c)(-) cystine/glutamate antiporter has been implicated in GSH-based chemoresistance because it mediates cellular uptake of cystine/cysteine for sustenance of intracellular GSH levels. Celastrol, isolated from a Chinese medicinal herb, is a novel heat shock protein 90 (Hsp90) inhibitor with potent anticancer activity against glioma in vitro and in vivo. In search of correlations between growth-inhibitory potency of celastrol in NCI-60 cell lines and microarray expression profiles of most known transporters, we found that expression of SLC7A11, the gene encoding the light chain subunit of x(c)(-), showed a strong negative correlation with celastrol activity. This novel gene-drug correlation was validated. In celastrol-resistant glioma cells that highly expressed SLC7A11, sensitivity to celastrol was consistently increased via treatment with x(c)(-) inhibitors, including glutamate, (S)-4-carboxyphenylglycine, sulfasalazine, and SLC7A11 small interfering RNA. The GSH synthesis inhibitor, buthionine sulfoximine, also increased celastrol sensitivity, whereas the GSH booster, N-acetylcysteine, suppressed its cytotoxicity. Furthermore, the glioma cell lines were dependent on x(c)(-)-mediated cystine uptake for viability, because cystine omission from the culture medium resulted in cell death and treatment with sulfasalazine depleted GSH levels and inhibited their growth. Combined treatment of glioma cells with sulfasalazine and celastrol led to chemosensitization, as suggested by increased celastrol-induced cell cycle arrest, apoptosis, and down-regulation of the Hsp90 client protein, epidermal growth factor receptor. These results indicate that the x(c)(-) transporter provides a useful target for glioma therapy. x(c)(-) inhibitors such as sulfasalazine, a Food and Drug Administration-approved drug, may be effective both as an anticancer drug and as an agent for sensitizing gliomas to celastrol.

Potential Therapies for Infectious Diseases Based on Targeting Immune Evasion Mechanisms That Pathogens Have in Common With Cancer Cells.

Many global infectious diseases are not well-controlled, underlining a critical need for new, more effective therapies. Pathogens and pathogen-infected host cells, like cancer cells, evade immune surveillance via immune evasion mechanisms. The present study indicates that pathogenic bacteria, endoparasites, and virus-infected host cells can have immune evasion mechanisms in common with cancers. These include entry into dormancy and metabolic reprogramming to aerobic glycolysis leading to excessive secretion of lactic acid and immobilization of local host immunity. The latter evasion tactic provides a therapeutic target for cancer, as shown by our recent finding that patient-derived cancer xenografts can be growth-arrested, without major host toxicity, by inhibiting their lactic acid secretion (as mediated by the MCT4 transporter)-with evidence of host immunity restoration. Accordingly, the multiplication of bacteria, endoparasites, and viruses that primarily depend on metabolic reprogramming to aerobic glycolysis for survival may be arrested using cancer treatment strategies that inhibit their lactic acid secretion. Immune evasion mechanisms shared by pathogens and cancer cells likely represent fundamental, evolutionarily-conserved mechanisms that may be particularly critical to their welfare. As such, their targeting may lead to novel therapies for infectious diseases.

The x(c)- cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases.

The x(c) (-) cystine/glutamate antiporter is a major plasma membrane transporter for the cellular uptake of cystine in exchange for intracellular glutamate. Its main functions in the body are mediation of cellular cystine uptake for synthesis of glutathione essential for cellular protection from oxidative stress and maintenance of a cystine:cysteine redox balance in the extracellular compartment. In the past decade it has become evident that the x(c) (-) transporter plays an important role in various aspects of cancer, including: (i) growth and progression of cancers that have a critical growth requirement for extracellular cystine/cysteine, (ii) glutathione-based drug resistance, (iii) excitotoxicity due to excessive release of glutamate, and (iv) uptake of herpesvirus 8, a causative agent of Kaposi's sarcoma. The x(c) (-) transporter also plays a role in certain CNS and eye diseases. This review focuses on the expression and function of the x(c) (-) transporter in cells and tissues with particular emphasis on its role in disease pathogenesis. The potential use of x(c) (-) inhibitors (e.g., sulfasalazine) for arresting tumor growth and/or sensitizing cancers is discussed.

Pre-clinical Models for Malignant Mesothelioma Research: From Chemical-Induced to Patient-Derived Cancer Xenografts.

Malignant mesothelioma (MM) is a rare disease often associated with environmental exposure to asbestos and other erionite fibers. MM has a long latency period prior to manifestation and a poor prognosis. The survival post-diagnosis is often less than a year. Although use of asbestos has been banned in the United States and many European countries, asbestos is still being used and extracted in many developing countries. Occupational exposure to asbestos, mining, and migration are reasons that we expect to continue to see growing incidence of mesothelioma in the coming decades. Despite improvements in survival achieved with multimodal therapies and cytoreductive surgeries, less morbid, more effective interventions are needed. Thus, identifying prognostic and predictive biomarkers for MM, and developing novel agents for targeted therapy, are key unmet needs in mesothelioma research and treatment. In this review, we discuss the evolution of pre-clinical model systems developed to study MM and emphasize the remarkable capability of patient-derived xenograft (PDX) MM models in expediting the pre-clinical development of novel therapeutic approaches. PDX disease model systems retain major characteristics of original malignancies with high fidelity, including molecular, histopathological and functional heterogeneities, and as such play major roles in translational research, drug development, and precision medicine.

Targeting MCT4 to reduce lactic acid secretion and glycolysis for treatment of neuroendocrine prostate cancer.

Development of neuroendocrine prostate cancer (NEPC) is emerging as a major problem in clinical management of advanced prostate cancer (PCa). As increasingly potent androgen receptor (AR)-targeting antiandrogens are more widely used, PCa transdifferentiation into AR-independent NEPC as a mechanism of treatment resistance becomes more common and precarious, since NEPC is a lethal PCa subtype urgently requiring effective therapy. Reprogrammed glucose metabolism of cancers, that is elevated aerobic glycolysis involving increased lactic acid production/secretion, plays a key role in multiple cancer-promoting processes and has been implicated in therapeutics development. Here, we examined NEPC glucose metabolism using our unique panel of patient-derived xenograft PCa models and patient tumors. By calculating metabolic pathway scores using gene expression data, we found that elevated glycolysis coupled to increased lactic acid production/secretion is an important metabolic feature of NEPC. Specific inhibition of expression of MCT4 (a plasma membrane lactic acid transporter) by antisense oligonucleotides led to reduced lactic acid secretion as well as reduced glucose metabolism and NEPC cell proliferation. Taken together, our results indicate that elevated glycolysis coupled to excessive MCT4-mediated lactic acid secretion is clinically relevant and functionally important to NEPC. Inhibition of MCT4 expression appears to be a promising therapeutic strategy for NEPC.

Heterochromatin Protein 1α Mediates Development and Aggressiveness of Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR.

Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant Prostate Cancer.

BACKGROUND: Although androgen deprivation therapy is initially effective in controlling growth of hormone-naive prostate cancers (HNPCs) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. OBJECTIVE: To identify CRPC driver genes that may provide new targets to enhance CRPC therapy. DESIGN, SETTING, AND PARTICIPANTS: Patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The functionality of a potential CRPC driver was validated via its knockdown in cultured prostate cancer cells; its clinical relevance was established using data from prostate cancer patient databases. RESULTS AND LIMITATIONS: Eighty genes were found to be significantly upregulated at the CRPC stage, while seven of them also showed elevated expression prior to CRPC development. Among the latter, growth factor receptor bound protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, elevated GRB10 expression in clinical prostate cancer samples correlated with more aggressive tumor types and poorer patient treatment outcome. GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. A positive correlation between AKT activity and GRB10 expression was also found in clinical cohorts. CONCLUSIONS: GRB10 acts as a driver of CRPC and sensitizes androgen receptor pathway inhibitors, and hence GRB10 targeting provides a novel therapeutic strategy for the disease. PATIENT SUMMARY: Development of castration-resistant prostate cancer (CRPC) is a major problem in the management of the disease. Using state-of-the-art patient-derived hormone-naive prostate cancer xenograft models, we found and validated the growth factor receptor bound protein 10 gene as a driver of CRPC, indicating that it may be used as a new molecular target to enhance current CRPC therapy.

Stromal Gene Expression is Predictive for Metastatic Primary Prostate Cancer.

BACKGROUND: Clinical grading systems using clinical features alongside nomograms lack precision in guiding treatment decisions in prostate cancer (PCa). There is a critical need for identification of biomarkers that can more accurately stratify patients with primary PCa. OBJECTIVE: To identify a robust prognostic signature to better distinguish indolent from aggressive prostate cancer (PCa). DESIGN, SETTING, AND PARTICIPANTS: To develop the signature, whole-genome and whole-transcriptome sequencing was conducted on five PCa patient-derived xenograft (PDX) models collected from independent foci of a single primary tumor and exhibiting variable metastatic phenotypes. Multiple independent clinical cohorts including an intermediate-risk cohort were used to validate the biomarkers. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The outcome measurement defining aggressive PCa was metastasis following radical prostatectomy. A generalized linear model with lasso regularization was used to build a 93-gene stroma-derived metastasis signature (SDMS). The SDMS association with metastasis was assessed using a Wilcoxon rank-sum test. Performance was evaluated using the area under the curve (AUC) for the receiver operating characteristic, and Kaplan-Meier curves. Univariable and multivariable regression models were used to compare the SDMS alongside clinicopathological variables and reported signatures. AUC was assessed to determine if SDMS is additive or synergistic to previously reported signatures. RESULTS AND LIMITATIONS: A close association between stromal gene expression and metastatic phenotype was observed. Accordingly, the SDMS was modeled and validated in multiple independent clinical cohorts. Patients with higher SDMS scores were found to have worse prognosis. Furthermore, SDMS was an independent prognostic factor, can stratify risk in intermediate-risk PCa, and can improve the performance of other previously reported signatures. CONCLUSIONS: Profiling of stromal gene expression led to development of an SDMS that was validated as independently prognostic for the metastatic potential of prostate tumors. PATIENT SUMMARY: Our stroma-derived metastasis signature can predict the metastatic potential of early stage disease and will strengthen decisions regarding selection of active surveillance versus surgery and/or radiation therapy for prostate cancer patients. Furthermore, profiling of stroma cells should be more consistent than profiling of diverse cellular populations of heterogeneous tumors.

Establishment in severe combined immunodeficiency mice of subrenal capsule xenografts and transplantable tumor lines from a variety of primary human lung cancers: potential models for studying tumor progression-related changes.

PURPOSE: Lung cancer is a biologically diverse disease and relevant models reflecting its diversity would facilitate the improvement of existing therapies. With a view to establishing such models, we developed and evaluated xenografts of a variety of human lung cancers. EXPERIMENTAL DESIGN: Using nonobese diabetic/severe combined immunodeficiency mice, subrenal capsule xenografts were generated from primary lung cancer tissue, including moderately and poorly differentiated squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, and carcinosarcoma. After 4 to 12 weeks, xenografts were harvested for serial transplantation and comparison with the original tissue via histologic, chromosomal, and cytogenetic analyses. RESULTS: Xenografts were successfully established. H&E staining showed that xenografts retained major histologic features of the original cancers. Immunohistochemistry and fluorescence in situ hybridization confirmed the human origin of the tumor cells and development in xenografts of murine supportive stroma. Four transplantable lines were developed from rapidly growing tumors (>5 generations), i.e., a small cell lung carcinoma, large cell undifferentiated carcinoma, pulmonary carcinosarcoma, and squamous cell carcinoma. Analyses including spectral karyotyping, comparative genomic hybridization, and fluorescence in situ hybridization, revealed that the xenografts were genetically similar to the original tumors, showing chromosomal abnormalities consistent with karyotypic changes reported for lung cancer. CONCLUSIONS: The subrenal capsule xenograft approach essentially provides a living tumor bank derived from patient material and a means for isolating and expanding specific cell populations. The transplantable tumor lines seem to provide good models for studying various aspects of tumor progression and a platform for developing novel therapeutic regimens, with the possibility of patient-tailored therapies.

Sulfasalazine-induced reduction of glutathione levels in breast cancer cells: enhancement of growth-inhibitory activity of Doxorubicin.

BACKGROUND: We previously showed that the anti-inflammatory drug, sulfasalazine (salicylazosulfapyridine, SASP), can arrest proliferation of MCF-7 and MDA-MB-231 mammary cancer cells by inhibiting uptake of cystine via the x(c-) cystine/glutamate antiporter. Here we examined SASP with regard to reduction of cellular glutathione (GSH) levels and drug efficacy-enhancing ability. METHODS: GSH levels were measured spectrophotometrically. Cellular drug retention was determined with 3H-labeled methotrexate, and drug efficacy with a colony formation assay. RESULTS: Incubation of the mammary cancer cells with SASP (0.3-0.5 mM) led to reduction of their GSH content in a time- and concentration-dependent manner. Similar to MK-571, a multidrug resistance-associated protein inhibitor, SASP increased intracellular accumulation of methotrexate. Preincubation of cells with SASP (0.3 mM) significantly enhanced the potency of the anticancer agent doxorubicin (2.5 nM). CONCLUSIONS: SASP-induced reduction of cellular GSH levels can lead to growth arrest of mammary cancer cells and enhancement of anticancer drug efficacy.

BIRC6 Targeting as Potential Therapy for Advanced, Enzalutamide-Resistant Prostate Cancer.

Purpose: Enzalutamide resistance has emerged as a major problem in the management of castration-resistant prostate cancer (CRPC). Research on therapy resistance of CRPCs has primarily focused on the androgen receptor pathway. In contrast, there is limited information on antiapoptotic mechanisms that may facilitate the treatment resistance. The inhibitor of apoptosis proteins (IAP) family is well recognized for its role in promoting treatment resistance of cancers by inhibiting drug-induced apoptosis. Here, we examined whether BIRC6, an IAP family member, has a role in enzalutamide resistance of CRPCs and could provide a therapeutic target for enzalutamide-resistant CRPC.Experimental Design: Use of enzalutamide-resistant CRPC models: (i) the transplantable, first high-fidelity LTL-313BR patient-derived enzalutamide-resistant CRPC tissue xenograft line showing primary enzalutamide resistance, (ii) MR42D and MR49F CRPC cells/xenografts showing acquired enzalutamide resistance. Specific BIRC6 downregulation in these models was produced using a BIRC6-targeting antisense oligonucleotide (ASO-6w2). Gene expression was determined by qRT-PCR and gene expression profiling. Molecular pathways associated with growth inhibition were assessed via gene enrichment analysis.Results: Of eight IAPs examined, BIRC6 was the only one showing elevated expression in both enzalutamide-resistant CRPC models. Treatment with ASO-6w2 markedly suppressed growth of LTL-313BR xenografts and increased tumor apoptosis without inducing major host toxicity. Pathway enrichment analysis indicated that GPCR and matrisome signaling were the most significantly altered pathways. Furthermore, ASO-6w2 inhibited expression of prosurvival genes that were upregulated in the LTL-313BR line.Conclusions:BIRC6 targeting inhibited the growth of enzalutamide-resistant CRPC models and may represent a new option for clinical treatment of advanced, enzalutamide-resistant prostate cancer. Clin Cancer Res; 23(6); 1542-51. ©2016 AACR.