Simon J. Hall

Autonomic Nerve Development Contributes to Prostate Cancer Progression

Introduction Cancer cells usurp the healthy tissue microenvironment to promote their survival, proliferation, and dissemination. The role of angiogenesis, the formation of new blood vessels, in solid tumor growth is well established. Whether neurogenesis, the formation of new nerve fibers, likewise contributes to tumor development and progression remains unclear. Here, studying mouse models and human tumor samples, we examined the role of the autonomic nervous system in prostate cancer growth and dissemination. The parasympathetic nervous system promotes prostate cancer dissemination in mice. Image shows bone metastases (arrowheads) detected by Na18F-PET scanning of Hi-Myc mice, a model of prostate cancer. Such metastases are not detected when the Hi-Myc mice are genetically deficient in muscarinic cholinergic receptor type 1, an essential signaling component of the parasympathetic branch of the autonomic nervous system. Methods To track tumor growth and dissemination, we studied (i) mice bearing PC-3 prostate tumor xenografts that expressed luciferase and (ii) transgenic mice expressing the c-Myc oncogene under the control of the probasin promoter (Hi-Myc mice), which develop prostatic intraepithelial neoplasia that progresses to invasive adenocarcinoma. Tumors were monitored by bioluminescence, positron emission tomography (PET), and histological analyses. Sympathetic (adrenergic) and parasympathetic (cholinergic) nerve functions were assessed using chemical or surgical neural ablation, pharmacological agonists or antagonists, and genetically engineered mice. We also determined the adrenergic and cholinergic nerve densities in radical prostatectomy tissues from a cohort of 43 patients with prostate cancer. Results Quantitative bioluminescence and immunofluorescence analyses, combined with histological examinations, revealed that sympathetic adrenergic nerve outgrowth was critical in the early phases of cancer development. Prostate tumor xenografts developed poorly in mice that had been pretreated by chemical or surgical sympathectomy of the prostate gland, or when stromal β2- and β3-adrenergic receptors were genetically deleted. Prostate tumors were also infiltrated by parasympathetic cholinergic fibers that promoted cancer dissemination. Cholinergic-induced tumor invasion and metastasis in mice were inhibited by pharmacological blockade or genetic disruption of the stromal type 1 muscarinic receptor. Quantitative confocal microscopy analysis of radical prostatectomy specimens from patients with low-risk (n = 30) or high-risk (n = 13) prostate adenocarcinoma revealed higher overall nerve densities in high-risk tumors relative to low-risk tumors. Adrenergic fibers were increased in normal prostate tissues surrounding the human tumors, whereas cholinergic fibers infiltrated the tumor tissue. Higher densities of adrenergic and cholinergic nerve fibers were associated with poor clinical outcome, including higher preoperative levels of prostate-specific antigen (PSA), extension beyond the prostatic capsule, and biochemical recurrence. Discussion These results suggest that the formation of new nerve fibers within and around prostate tumors can alter tumor behavior. The autonomic nervous system appears to exert dual functions in prostate cancer: Sympathetic neonerves promote early stages of tumorigenesis, whereas parasympathetic nerve fibers promote cancer dissemination. Conceivably, drugs targeting both branches of the autonomic nervous system could provide therapeutic benefit. Cancer Hits a Nerve Solid tumors sculpt their microenvironment to maximize their growth and metastatic potential. This concept is illustrated most famously by tumor angiogenesis, a process whereby tumors induce the growth of new blood vessels to boost their supply of oxygen and blood-borne nutrients. Magnon et al. (p. 10.1126/science.1236361; see the Perspective by Isaacs) now highlight the important contribution made by another microenvironmental component—developing autonomic nerve fibers—to tumor growth and metastasis. In mouse models of prostate cancer, surgical or chemical destruction of sympathetic nerves prevented early-stage growth of tumors, whereas pharmacological inhibition of parasympathetic nerves inhibited tumor dissemination. In a small study of human prostate cancer specimens, the presence of a high density of nerve fibers in and around the tumor tissue was found to correlate with poor clinical outcome. These results raise the possibility that drugs targeting the autonomic nervous system may have therapeutic potential for prostate cancer. Prostate cancer is more aggressive when certain types of nerves form near and within the tumor. [Also see Perspective by Isaacs] Nerves are a common feature of the microenvironment, but their role in tumor growth and progression remains unclear. We found that the formation of autonomic nerve fibers in the prostate gland regulates prostate cancer development and dissemination in mouse models. The early phases of tumor development were prevented by chemical or surgical sympathectomy and by genetic deletion of stromal β2- and β3-adrenergic receptors. Tumors were also infiltrated by parasympathetic cholinergic fibers that promoted cancer dissemination. Cholinergic-induced tumor invasion and metastasis were inhibited by pharmacological blockade or genetic disruption of the stromal type 1 muscarinic receptor, leading to improved survival of the mice. A retrospective blinded analysis of prostate adenocarcinoma specimens from 43 patients revealed that the densities of sympathetic and parasympathetic nerve fibers in tumor and surrounding normal tissue, respectively, were associated with poor clinical outcomes. These findings may lead to novel therapeutic approaches for prostate cancer.

C-terminal Hsp-interacting protein slows androgen receptor synthesis and reduces its rate of degradation

The androgen receptor (AR) is a member of the nuclear receptor superfamily that requires the action of molecular chaperones for folding and hormone binding. C-terminal Hsp-interacting protein (Chip) is a cochaperone that interacts with Hsp70 and Hsp90 molecular chaperones via a tetratricopeptide domain and inhibits chaperone-dependent protein folding in vitro. Chip also stimulates protein degradation by acting as an E3 ubiquitin ligase via a modified ring finger domain called a U box. We analyzed whether Chip affected AR levels using a transient transfection strategy. Chip overexpression led to a large decrease in AR steady state levels and increased levels of AR ubiquitinylation. However, Chip effects were not fully reversed by proteasome inhibitors, suggesting that mechanisms alternative to or in addition to proteasome-mediated degradation were involved. This hypothesis was supported by the finding that Chip overexpression reduced the rate of AR degradation, consistent with an effect on AR folding, perhaps leading to aggregation. The possibility that Chip affected AR folding was further supported by the finding that the effects of exogenous Chip were reproduced by a mutant lacking the U box. These results are discussed in terms of the role played by molecular chaperones in AR biogenesis.

Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3zeta chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.

Randomized trial of autologous cellular immunotherapy with sipuleucel-T in androgen-dependent prostate cancer

Purpose: Sipuleucel-T, an autologous cellular immunotherapy, was investigated in a randomized, double-blind, controlled trial to determine its biologic activity in androgen-dependent prostate cancer (ADPC). Experimental Design: Patients with prostate cancer detectable by serum prostate-specific antigen (PSA) following radical prostatectomy received 3 to 4 months of androgen suppression therapy, and were then randomized (2:1) to receive sipuleucel-T (n = 117) or control (n = 59). The primary endpoint was time to biochemical failure (BF) defined as serum PSA ≥ 3.0 ng/mL. PSA doubling time (PSADT), time to distant failure, immune response, and safety were also evaluated. Results: Median time to BF was 18.0 months for sipuleucel-T and 15.4 months for control (HR = 0.936, P = 0.737). Sipuleucel-T patients had a 48% increase in PSADT following testosterone recovery (155 vs. 105 days, P = 0.038). With only 16% of patients having developed distant failure, the treatment effect favored sipuleucel-T (HR = 0.728, P = 0.421). The most frequent adverse events in sipuleucel-T patients were fatigue, chills, and pyrexia. Immune responses to the immunizing antigen were greater in sipuleucel-T patients at Weeks 4 and 13 (P < 0.001, all) and were sustained prior to boosting as measured in a subset of patients a median of 22.6 months (range: 14.3–67.3 months) following randomization. Conclusions: No significant difference in time to BF could be shown. The finding of increased PSADT in the sipuleucel-T arm is consistent with its biologic activity in ADPC. Long-term follow-up will be necessary to determine if clinically important events, such as distant failure, are affected by therapy. Treatment was generally well tolerated. Clin Cancer Res; 17(13); 4558–67. ©2011 AACR.

KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis

Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1-overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target.

Humoral Immune Response against Nontargeted Tumor Antigens after Treatment with Sipuleucel-T and Its Association with Improved Clinical Outcome

Purpose: Antitumor activity of cancer immunotherapies may elicit immune responses to nontargeted (secondary) tumor antigens, or antigen spread. We evaluated humoral antigen spread after treatment with sipuleucel-T, an immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC), designed to target prostatic acid phosphatase (PAP; primary antigen). Experimental Design: Serum samples from patients with mCRPC enrolled in the placebo-controlled phase III IMPACT study (evaluable n = 142) were used to assess humoral antigen spread after treatment with sipuleucel-T. Immunoglobulin G (IgG) responses to self-antigens (including tumor antigens) were surveyed using protein microarrays and confirmed using Luminex xMAP. IgG responses were subsequently validated in ProACT (n = 33), an independent phase II study of sipuleucel-T. Association of IgG responses with overall survival (OS) was assessed using multivariate Cox models adjusted for baseline prostate-specific antigen (PSA) and lactate dehydrogenase levels. Results: In patients from IMPACT and ProACT, levels of IgG against multiple secondary antigens, including PSA, KLK2/hK2, K-Ras, E-Ras, LGALS8/PCTA-1/galectin-8, and LGALS3/galectin-3, were elevated after treatment with sipuleucel-T (P < 0.01), but not control. IgG responses (≥2-fold elevation posttreatment) occurred in ≥25% of patients, appeared by 2 weeks after sipuleucel-T treatment, and persisted for up to 6 months. IgG responses to PSA and LGALS3 were associated with improved OS in sipuleucel-T–treated patients from IMPACT (P ≤ 0.05). Conclusions: Sipuleucel-T induced humoral antigen spread in patients with mCRPC. IgG responses were associated with improved OS in IMPACT. The methods and results reported may identify pharmacodynamic biomarkers of clinical outcome after sipuleucel-T treatment, and help in clinical assessments of other cancer immunotherapies. Clin Cancer Res; 21(16); 3619–30. ©2015 AACR. See related commentary by Hellstrom and Hellstrom, p. 3581

Outcomes for patients with high―grade prostate cancer treated with a combination of brachytherapy, external beam radiotherapy and hormonal therapy

To assess the outcomes for patients with Gleason score 8–10 prostate cancer treated with brachytherapy, external beam radiotherapy (EBRT) and hormonal therapy (HT).

Prodrug activation gene therapy and external beam irradiation in the treatment of prostate cancer

OBJECTIVES Gene therapy may represent a new avenue for the development of multimodal treatment for men with locally advanced prostate cancer. This study explores the potential benefits of combining adenovirus-mediated (ADV) herpes simplex virus thymidine kinase gene (HSV-tk) transduction and ganciclovir (GCV) therapy with external beam radiation therapy (XRT) to enhance the therapeutic efficacy of each treatment alone. METHODS ADV/HSV-tk-transduced mouse prostate cancer cells, RM-1, were irradiated as single-cell suspensions at escalating doses in a cesium source (4.4 Gy/min). HSV-tk-expressing cells were randomized to receive varying doses and varying chronologies of GCV therapy in relation to XRT to fully evaluate potential cooperative activities. End points were determined in a clonogenic assay by counting colonies with greater than 50 cells 7 days after replating. The potential role of apoptosis as a mediator of enhanced cell killing was addressed by a TUNEL assay 12 and 24 hours after therapy. RESULTS Neither ADV infection nor GCV alone affected XRT killing. However, the combination of ADV/HSV-tk+GCV plus XRT maintained the 1 log of cell kill from gene therapy alone through escalating doses of radiation. Radiation sensitization was noted at higher doses of radiation (8.8 Gy or more). Although decreasing the GCV dose had a profound negative influence on HSV-tk+GCV-mediated killing, combination therapy continued to maintain the degree of HSV-tk+GCV killing through escalating doses of XRT in an additive fashion but did not result in radiosensitization. Changing the chronology of GCV exposure in relation to XRT did not significantly alter the additive activities of combination therapy. Studies of apoptosis noted a doubling of apoptotic activity with HSV-tk+GCV compared with HSV-tk+PBS with or without XRT. However, there was no significant change in apoptotic activity in combination therapy over HSV-tk+GCV alone within the 24-hour period after GCV exposure. CONCLUSIONS The combination of ADV/HSV-tk+GCV and XRT appears to result in at least additive, and with higher doses of radiation, synergistic killing activities, indicating a potential usefulness of this treatment strategy for patients with prostate cancer.

Muscle Invasive Bladder Cancer: Examining Survivor Burden and Unmet Needs

PURPOSE Although improvements in perioperative care have decreased surgical morbidity after radical cystectomy for muscle invasive bladder cancer, treatment side effects still have a negative impact on patient quality of life. We examined unmet patient needs along the illness trajectory. MATERIALS AND METHODS A total of 30 patients (26.7% women) treated with cystectomy and urinary diversion for muscle invasive bladder cancer participated in the study. Patients were recruited from the Department of Urology at Mount Sinai and through advertisements on the Bladder Cancer Advocacy Network (BCAN) website between December 2011 and September 2012. Data were collected at individual interviews, which were audiotaped and transcribed. Transcribed data were quantitatively analyzed to explore key unmet needs. RESULTS At diagnosis unmet informational needs were predominant, consisting of insufficient discussion of certain topics, including urinary diversion options and their side effects, self-care, the recovery process and medical insurance. Unmet psychological needs related to depression, and worries about changes in body image and sexual function were reported. Postoperative unmet needs revolved around medical needs (eg pain and bowel dysfunction) and instrumental needs (eg need of support for stomal appliances, catheters and incontinence). During survivorship (ie 6 to 72 months postoperatively) unmet needs centered around psychological support (ie depression, poor body image and sexual dysfunction) and instrumental support (eg difficulty adjusting to changes in daily living). CONCLUSIONS Meeting patient needs is imperative to ensure adequate patient involvement in health care and enhance postoperative quality of life. An effective support provision plan should follow changes in patient needs.

The Emerging Role of Circulating Tumor Cell Detection in Genitourinary Cancer

PURPOSE Circulating tumor cells are malignant cells in peripheral blood that originate from primary tumors or metastatic sites. The heterogeneous natural history and propensity for recurrence in prostate, bladder and kidney cancers are well suited for improved individualization of care using circulating tumor cells. The potential clinical applications of circulating tumor cells include early diagnosis, disease prediction and prognosis, and selection of appropriate therapies. MATERIALS AND METHODS The PubMed® and Web of Science® databases were searched using the key words circulating tumor cells, CTC, prostate, kidney, bladder, renal cell carcinoma and transitional cell carcinoma. Relevant articles and references from 1994 to 2011 were reviewed for data on the detection and significance of circulating tumor cells in genitourinary cancer. RESULTS Technical challenges have previously limited the widespread introduction of circulating tumor cell detection in routine clinical care. Recently novel platforms were introduced to detect these cells that offer the promise of overcoming these limitations. We reviewed the current state of circulating tumor cell capture technologies and their clinical applications for genitourinary cancers. CONCLUSIONS In genitourinary cancer circulating tumor cell enumeration has been useful for prognosis in patients with castration resistant prostate cancer. Soon characterizing individual circulating tumor cells in blood will serve as a noninvasive real-time liquid biopsy to monitor molecular changes in cancer, allowing clinicians to custom tailor treatment strategies. Circulating tumor cells will serve as a treatment response biomarker. Finally, circulating tumor cell detection promises to assist in the early detection of clinically localized cancers, facilitating curative therapy.