David Pook

A preclinical xenograft model of prostate cancer using human tumors

Most cases of prostate cancer are now diagnosed as moderate-grade localized disease. These tumor specimens are important tools in the discovery and translation of prostate cancer research; however, unlike more advanced tumors, they are notoriously difficult to grow in the laboratory. We developed a system for efficiently xenografting localized human prostate cancer tissue, and we adapted this protocol to study the interactions between the specific subsets of epithelial and stromal cells. Fresh prostate tissues or isolated epithelial cells are recombined with mouse seminal vesicle mesenchyme (SVM) and grafted under the renal capsule of immunodeficient mice for optimum growth and survival. Alternatively, mouse mesenchyme can be replaced with human prostate fibroblasts in order to determine their contribution to tumor progression. Grafts can be grown for several months to determine the effectiveness of novel therapeutic compounds when administered to host mice, thereby paving the way for personalizing the treatment of individual prostate cancers.

A Preclinical Xenograft Model Identifies Castration-Tolerant Cancer-Repopulating Cells in Localized Prostate Tumors

This study uses a preclinical xenograft model to reveal prostate cancer cells that exist in untreated localized disease, survive androgen withdrawal, and are potential therapeutic targets. The Enemy Within Prostate cancer is one of the most common types of cancer in men. In advanced stages, it is typically treated with medications that mimic castration, depriving the tumor of androgen stimulation. Unfortunately, these cancers eventually become castration-resistant and begin to grow even in the absence of hormonal input. What isn’t known is how these cancer cells develop the ability to survive androgen deprivation, and whether some types of stem-like castration-resistant cells are already present in prostate cancer from early stages or evolve later during the course of treatment. Now, Toivanen and colleagues shed some light on this mystery, with a report of castration-tolerant cells derived from early localized tumors that had not yet been exposed to anti-androgen therapy. The authors used primary prostate tumors from 12 men with localized cancer, implanting them in a mouse xenograft model to study the effects of androgen deprivation on the tumors’ survival. Castration of the host mice led to rapid regression, but not disappearance of the tumors. Even after a prolonged period of castration (4 weeks), some residual tumor foci persisted. When testosterone stimulation was restored in the host animals, these residual cells rebounded, regenerating masses that were histologically similar to the original tumors. This work by Toivanen et al. indicates that some prostate cancer cells can survive castration and later repopulate the tumor when androgen stimulation is available. Thus far, there is no indication that these castration-tolerant cells can proliferate in the absence of androgens, unlike the cells found in more advanced “castration-resistant” prostate cancer. Additional work will be needed to clarify whether these might be a type of prostate cancer stem cells, and what makes them different from the population of “androgen-sensitive” cancer cells that do not survive androgen depletion. Although there are many questions that must still be answered about the biology of these castration-tolerant cells, this work raises the intriguing possibility that we may eventually be able to specifically target and eradicate them, thus preventing prostate cancer recurrence in patients. A lack of clinically relevant experimental models of human prostate cancer hampers evaluation of potential therapeutic agents. Currently, androgen deprivation therapy is the gold standard treatment for advanced prostate cancer, but inevitably, a subpopulation of cancer cells survives and repopulates the tumor. Tumor cells that survive androgen withdrawal are critical therapeutic targets for more effective treatments, but current model systems cannot determine when they arise in disease progression and are unable to recapitulate variable patient response to treatment. A model system was developed in which stromal-supported xenografts from multiple patients with early-stage localized disease can be tested for response to castration. The histopathology of these xenografts mimicked the original tumors, and short-term host castration resulted in reduced proliferation and increased apoptosis in tumor cells. After 4 weeks of castration, residual populations of quiescent, stem-like tumor cells remained. Without subsequent treatment, these residual cells displayed regenerative potential, because testosterone readministration resulted in emergence of rapidly proliferating tumors. Therefore, this model may be useful for revealing potential cellular targets in prostate cancer, which exist before the onset of aggressive incurable disease. Specific eradication of these regenerative tumor cells that survive castration could then confer survival benefits for patients.

A bioengineered microenvironment to quantitatively measure the tumorigenic properties of cancer-associated fibroblasts in human prostate cancer

Stromal-epithelial cell interactions play an important role in cancer and the tumor stroma is regarded as a therapeutic target. In vivo xenografting is commonly used to study cellular interactions not mimicked or quantified in conventional 2D culture systems. To interrogate the effects of tumor stroma (cancer-associated fibroblasts or CAFs) on epithelia, we created a bioengineered microenvironment using human prostatic tissues. Patient-matched CAFs and non-malignant prostatic fibroblasts (NPFs) from men with moderate (Gleason 7) and aggressive (Gleason 8-9 or castrate-resistant) prostate cancer were cultured with non-tumorigenic BPH-1 epithelial cells. Changes in the morphology, motility and phenotype of BPH-1 cells in response to CAFs and NPFs were analyzed using immunofluorescence and quantitative cell morphometric analyses. The matrix protein gene expression of CAFs, with proven tumorigenicity in vivo, had a significantly different gene expression profile of matrix proteins compared to patient matched NPFs. In co-culture with CAFs (but not NPFs), BPH-1 cells had a more invasive, elongated phenotype with increased motility and a more directed pattern of cell migration. CAFs from more aggressive tumors (Gleason 8-9 or CRPC) were not quantitatively different to moderate grade CAFs. Overall, our bioengineered microenvironment provides a novel 3D in vitro platform to systematically investigate the effects of tumor stroma on prostate cancer progression.

Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.

Breaking through a roadblock in prostate cancer research: An update on human model systems

Prostate cancer is a prevalent disease that affects the aging male population. Whilst there have been significant advances of our biological understanding of the disease, clinical translation of promising agents continues to lag behind. In part, this is due to a paucity of relevant experimental and pre-clinical models required to further develop effective prevention and therapeutic strategies. Genetically modified cell lines fail to entirely represent the genetic and molecular diversity of primary human specimens, particularly from localised disease. Furthermore, primary prostate cancer tissues are extremely difficult to grow in the laboratory and virtually all human models, whether they grow as xenografts in immune-deficient animals or as cell cultures, are genetically modified by the investigator or derived from patients with advanced metastatic disease. In this review, we discuss the latest advances and improvements to current methods of xenografting human primary prostate cancer, and their potential application to translational research.

Patient-derived Xenografts Reveal that Intraductal Carcinoma of the Prostate Is a Prominent Pathology in BRCA2 Mutation Carriers with Prostate Cancer and Correlates with Poor Prognosis

BACKGROUND Intraductal carcinoma of the prostate (IDC-P) is a distinct clinicopathologic entity associated with aggressive prostate cancer (PCa). PCa patients carrying a breast cancer 2, early onset (BRCA2) germline mutation exhibit highly aggressive tumours with poor prognosis. OBJECTIVE To investigate the presence and implications of IDC-P in men with a strong family history of PCa who either carry a BRCA2 pathogenic mutation or do not carry the mutation (BRCAX). DESIGN, SETTING, AND PARTICIPANTS Patient-derived xenografts (PDXs) were generated from three germline BRCA2 mutation carriers and one BRCAX patient. Specimens were examined for histologic evidence of IDC-P. Whole-genome copy number analysis (WG-CNA) was performed on IDC-P from a primary and a matched PDX specimen. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The incidence of IDC-P and association with overall survival for BRCA2 and BRCAX patients were determined using Kaplan-Meier analysis. RESULTS AND LIMITATIONS PDXs from BRCA2 tumours showed increased incidence of IDC-P compared with sporadic PCa (p=0.015). WG-CNA confirmed that the genetic profile of IDC-P from a matched (primary and PDX) BRCA2 tumour was similar. The incidence of IDC-P was significantly increased in BRCA2 carriers (42%, n=33, p=0.004) but not in BRCAX patients (25.8%, n=62, p=0.102) when both groups were compared with sporadic cases (9%, n=32). BRCA2 carriers and BRCAX patients with IDC-P had significantly worse overall and PCa-specific survival compared with BRCA2 carriers and BRCAX patients without IDC-P (hazard ratio [HR]: 16.9, p=0.0064 and HR: 3.57, p=0.0086, respectively). CONCLUSIONS PDXs revealed IDC-P in patients with germline BRCA2 mutations or BRCAX classification, identifying aggressive tumours with poor survival even when the stage and grade of cancer at diagnosis were similar. Further studies of the prognostic significance of IDC-P in sporadic PCa are warranted. PATIENT SUMMARY Intraductal carcinoma of the prostate is common in patients with familial prostate cancer and is associated with poor outcomes. This finding affects genetic counselling and identifies patients in whom earlier multimodality treatment may be required.

Motesanib diphosphate (AMG 706), an oral angiogenesis inhibitor, demonstrates clinical efficacy in advanced thymoma

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A pro-tumourigenic loop at the human prostate tumour interface orchestrated by oestrogen, CXCL12 and mast cell recruitment.

Prostate cancer is hormone‐dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro‐tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma‐associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERβ, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERβ transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen‐driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4‐dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface. Copyright

Establishment of primary patient-derived xenografts of palliative TURP specimens to study castrate-resistant prostate cancer.

Fresh patient specimens of castrate‐resistant prostate cancer (CRPC) are invaluable for studying tumor heterogeneity and responses to current treatments. They can be used for primary patient‐derived xenografts (PDXs) or serially transplantable PDXs, but only a small proportion of samples grow successfully. To improve the efficiency and quality of PDXs, we investigated the factors that determine the initial engraftment of patient tissues derived from TURP specimens.

Rare synchronous metastases of renal cell carcinoma

Renal cell carcinoma can metastasize to any region of the body. We review a patient who presents fourteen years after initial resection of the primary tumor with distant metastatic disease. This included spread to the bladder and penis that manifested as frank haematuria and malignant priapism respectively. We discuss the mechanism of spread and the management options available.