Amy Strasner

Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL–RANK signalling

Inflammatory mechanisms influence tumorigenesis and metastatic progression even in cancers whose aetiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. For instance, prostate cancer metastasis is associated with the infiltration of lymphocytes into advanced tumours and the upregulation of two tumour-necrosis-factor family members: receptor activator of nuclear factor-κB (RANK) ligand (RANKL) and lymphotoxin. But the source of RANKL and its role in metastasis have not been established. RANKL and its receptor RANK control the proliferation of mammary lobuloalveolar cells during pregnancy through inhibitor of nuclear factor-κB (IκB) kinase-α (IKK-α), a protein kinase that is needed for the self-renewal of mammary cancer progenitors and for prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKK-α are also involved in mammary/breast cancer metastasis. Indeed, RANK signalling in mammary carcinoma cells that overexpress the proto-oncogene Erbb2 (also known as Neu), which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of Erbb2-transformed carcinoma cells also required CD4+CD25+ T cells, whose major pro-metastatic function was RANKL production. Most RANKL-producing T cells expressed forkhead box P3 (FOXP3), a transcription factor produced by regulatory T cells, and were located next to smooth muscle actin (SMA)+ stromal cells in mouse and human breast cancers. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK+ human breast cancer cells. These results are consistent with the adverse impact of tumour-infiltrating CD4+ or FOXP3+ T cells on human breast cancer prognosis and suggest that the targeting of RANKL–RANK can be used in conjunction with the therapeutic elimination of primary breast tumours to prevent recurrent metastatic disease.

Immunosuppressive plasma cells impede T cell-dependent immunogenic chemotherapy

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8+ cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFβ receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

Tumor infiltrating B-cells are increased in prostate cancer tissue

BackgroundThe presence of increased B-cell tumor infiltrating lymphocytes (TILs) was seen in mouse prostate cancer (PCa) but has not been fully documented in human PCa. We, therefore, investigated the density of infiltrating B cells within human PCa utilizing a quantitative computational method.MethodsArchived radical prostatectomy specimens from 53 patients with known clinical outcome and D’Amico risk category were obtained and immunohistochemically (IHC) stained for the B cell marker, CD20. Slides were reviewed by a genitourinary pathologist who manually delineated the tumoral regions of PCa. Slides were digitally scanned and a computer algorithm quantified the area of CD20 stained B-cells as a measure of B cell density within the outlined regions of prostate cancer (intra-tumoral region), versus extra-tumoral prostate tissue. Correlations were analyzed between B-cell density and demographic and clinical variables, including D’Amico risk groups and disease recurrence.ResultsFor the entire cohort, the mean intra-tumoral B cell density was higher (3.22 SE = 0.29) than in the extra-tumoral region of each prostatectomy section (2.24, SE = 0.19) (paired t test; P < 0.001). When analyzed according to D’Amico risk group, the intra-tumoral B cell infiltration in low risk (0.0377 vs. 0.0246; p = 0.151) and intermediate risk (0.0260 vs. 0.0214; p = 0.579) patient prostatectomy specimens did not show significantly more B-cells within the PCa tumor. However, patient specimens from the high-risk group (0.0301 vs. 0.0197; p < 0.001) and from those who eventually had PCa recurrence or progression (0.0343 vs. 0.0246; p = 0.019) did show significantly more intra-tumoral CD20+ B-cell staining. Extent of B-cell infiltration in the prostatectomy specimens did not correlate with any other clinical parameters.ConclusionsOur study shows that higher B-cell infiltration was present within the intra-tumoral PCa regions compared to the extra-tumoral benign prostate tissue regions in prostatectomy sections. For this study we developed a new method to measure B-cells using computer-assisted digitized image analysis. Accurate, consistent quantitation of B-cells in prostatectomy specimens is essential for future clinical trials evaluating the effect of B cell ablating antibodies. The interaction of B-cells and PCa may serve as the basis for new therapeutic targets.

A NIK-IKKα Module Expands ErbB2-Induced Tumor-Initiating Cells by Stimulating Nuclear Export of p27/Kip1

IκB kinase α (IKKα) activity is required for ErbB2-induced mammary tumorigenesis. Here, we show that IKKα and its activator, NF-κB-inducing kinase (NIK), support the expansion of tumor-initiating cells (TICs) that copurify with a CD24(med)CD49f(hi) population from premalignant ErbB2-expressing mammary glands. Upon activation, IKKα enters the nucleus, phosphorylates the cyclin-dependent kinase (CDK) inhibitor p27/Kip1, and stimulates its nuclear export or exclusion. Reduced p27 expression rescues mammary tumorigenesis in mice deficient in IKKα kinase activity and restores TIC self-renewal. IKKα is also likely to be involved in human breast cancer, where its expression shows an inverse correlation with metastasis-free survival, and its presence in the nucleus of invasive ductal carcinomas (IDCs) is associated with decreased nuclear p27 abundance.

Immune Infiltration and Prostate Cancer

It is becoming increasingly clear that inflammation influences prostate cancer (PCa) development and that immune cells are among the primary drivers of this effect. This information has launched numerous clinical trials testing immunotherapy drugs in PCa patients. The results of these studies are promising but have yet to generate a complete response. Importantly, the precise immune profile that determines clinical outcome remains unresolved. Individual immune cell types are divided into various functional subsets whose effects on tumor development may differ depending on their particular phenotype and functional status, which is often shaped by the tumor microenvironment. Thus, this review aims to examine the current knowledge regarding the role of inflammation and specific immune cell types in mediating PCa progression to assist in directing and optimizing immunotherapy targets, regimens, and responses and to uncover areas in which further research is needed. Finally, a summary of ongoing immunotherapy clinical trials in PCa is provided.

An IKKα–E2F1–BMI1 cascade activated by infiltrating B cells controls prostate regeneration and tumor recurrence

Androgen-deprived prostate cancer (PCa) is infiltrated by B lymphocytes that produce cytokines that activate IκB kinase α (IKKα) to accelerate the emergence of castration-resistant tumors. We now demonstrate that infiltrating B lymphocytes and IKKα are also required for androgen-dependent expansion of epithelial progenitors responsible for prostate regeneration. In these cells and in PCa cells, IKKα phosphorylates transcription factor E2F1 on a site that promotes its nuclear translocation, association with the coactivator CBP, and recruitment to critical genomic targets that include Bmi1, a key regulator of normal and cancerous prostate stem cell renewal. The IKKα-BMI1 pathway is also activated in human PCa.

PCSD1, a new patient-derived model of bone metastatic prostate cancer, is castrate-resistant in the bone-niche.

IntroductionProstate cancer bone metastasis occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with therapy resistance and rapid decline. Androgen deprivation therapy (ADT) is standard of care for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to ADT. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. Here we report the castrate resistant growth in the bone niche of PCSD1, a patient-derived intra-femoral xenograft model of prostate bone metastatic cancer treated with the anti-androgen, bicalutamide.MethodsPCSD1 bone-niche model was derived from a human prostate cancer femoral metastasis resected during hemiarthroplasty and serially transplanted into Rag2−/−;γc−/− mice intra-femorally (IF) or sub-cutaneously (SC). At 5 weeks post-transplantation mice received bicalutamide or vehicle control for 18 days. Tumor growth of PCSD1 was measured with calipers. PSA expression in PCSD1 xenograft tumors was determined using quantitative RT-PCR and immunohistochemistry. Expression of AR and PSMA, were also determined with qPCR.ResultsPCSD1 xenograft tumor growth capacity was 24 fold greater in the bone (intra-femoral, IF) than in the soft tissue (sub-cutaneous, SC) microenvironment. Treatment with the anti-androgen, bicalutamide, inhibited tumor growth in the sub-cutaneous transplantation site. However, bicalutamide was ineffective in suppressing PCSD1 tumor growth in the bone-niche. Nevertheless, bicalutamide treatment of intra-femoral tumors significantly reduced PSA expression (p < =0.008) and increased AR (p < =0.032) relative to control.ConclusionsPCSD1 tumors were castrate resistant when growing in the bone-niche compared to soft tissue. Bicalutamide had little effect on reducing tumor burden in the bone yet still decreased tumor PSA expression and increased AR expression, thus, this model closely recapitulated castrate-resistant, human prostate cancer bone metastatic disease. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study bone metastatic disease and for pre-clinical drug development of novel therapies for inhibiting therapy resistant prostate cancer growth in the bone-niche.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer

Objective Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. Methods Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2−/−γc−/− male mice. Results Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. Conclusion PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Abstract A43: Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer

Prostate cancer metastasis to bone occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with disease progression, therapy resistance, poor prognosis, and rapid decline. Androgen ablation therapy is standard of care for advanced prostate cancer, however, the role of androgens in bone metastatic prostate cancer is not understood. The effects of anti-androgens as seen on bone scans can also be inconsistent with the biochemical PSA response. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. It is essential to understand the unique interaction of prostate cancer with the bone environment and to develop novel therapies that target these pathways. Here we report the development of novel patient-derived intra-femoral xenograft models of prostate bone metastatic cancer. METHODS: Surgical prostate cancer bone metastasis specimens were transplanted by direct injection into the femurs of Rag2-/-γc-/- mice or sub-cutaneously into the right flank. Patient-derived xenograft (PDX) tumors that grew out were analyzed for prostate cancer biomarker expression using quantitative RT-PCR and immunohistochemistry. Bone lesion formation was measured using micro-computed tomography (μCT). RESULTS: Prostate cancer surgical bone metastasis specimens have been collected from which we have established new serially transplantable, prostate cancer bone metastasis xenograft models – PCSD1, PCSD4 and PCSD5. PCSD1 (Prostate Cancer San Diego 1) was molecularly characterized as advanced, luminal epithelial-type prostate cancer. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely mimicked those of the patient. Treatment with the anti-androgen, bicalutamide, did not inhibit intra-femoral PCSD1 xenograft growth although there was a decrease in PSA as seen in some patients treated with anti-androgen who had discordant PSA and bone scan tests. CONCLUSION: PCSD1, PCSD4 and PCSD5 are new patient-derived prostate cancer bone metastasis-derived xenograft models. PCSD1 xenograft model closely recapitulates the mixed osteolytic/osteoblastic bone metastatic lesions seen in patients, and we are using it to develop novel therapies for inhibiting prostate cancer growth in the bone-niche. Citation Format: Christina Jamieson, Christina Wu, Amy Strasner, Jason R. Woo, Michelle Muldong, Young B. Jeong, Michael A. Liss, Omer Raheem, Tomonori Yamaguchi, Heather Leu, Deborah Marshall, Sheldon Morris, Nicholas A. Cacalano, Koichi Masuda, Catriona H.M. Jamieson, Anna A. Kulidjian, Christopher J. Kane. Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A43.

Abstract 445: Fibroblast-recruited, tumor-infiltrating CD4+ T cells stimulate mammary cancer metastasis through RANKL-RANK signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Inflammatory mechanisms influence tumor development and metastatic progression. Of interest is the role of such mechanisms in metastatic spread of tumors whose etiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. We found that prostate cancer metastasis is associated with lymphocyte infiltration into advanced tumors and elevated expression of the tumor necrosis factor (TNF) family members receptor activator of NF-κB (RANK) ligand (RANKL) and lymphotoxin (LT). But the source of RANKL and its role in metastasis were not established. RANKL and its receptor RANK control proliferation of mammary lobuloalveolar cells during pregnancy through activation of IκB kinase α(IKKα), a protein kinase that is required for self-renewal of mammary cancer progenitors and prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKKα are also involved in mammary/breast cancer metastasis. Indeed, RANK signaling in mammary carcinoma cells that overexpress the ErbB2 (c-Neu) proto-oncogene, which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of ErbB2-transformed carcinoma cells was also dependent on CD4+CD25+ T cells, whose major pro-metastatic function appeared to be RANKL production. RANKL-producing T cells were mainly FoxP3+ and found in close proximity to smooth muscle actin (SMA)-positive stromal cells in mouse and human breast cancers. The T cell-dependence of pulmonary metastasis was replaced by administration of exogenous RANKL, a procedure that also stimulated pulmonary metastasis of RANK-positive human breast carcinoma cells. These results are consistent with the adverse prognostic impact of tumor-infiltrating CD4+ or FoxP3+ T cells on human breast cancer, and suggest that targeting of RANKL-RANK signaling can be used in conjunction with other therapies to prevent subsequent metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2011-445