Mitchell G. Lawrence

Epithelial-mesenchymal and mesenchymal : Epithelial transitions in carcinoma progression

Like a set of bookends, cellular, molecular, and genetic changes of the beginnings of life mirror those of one of the most common cause of death—metastatic cancer. Epithelial to mesenchymal transition (EMT) is an important change in cell phenotype which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized by Elizabeth Hay in the early to mid 1980s to be a central process in early embryonic morphogenesis. Reversals of these changes, termed mesenchymal to epithelial transitions (METs), also occur and are important in tissue construction in normal development. Over the last decade, evidence has mounted for EMT as the means through which solid tissue epithelial cancers invade and metastasize. However, demonstrating this potentially rapid and transient process in vivo has proven difficult and data connecting the relevance of this process to tumor progression is still somewhat limited and controversial. Evidence for an important role of MET in the development of clinically overt metastases is starting to accumulate, and model systems have been developed. This review details recent advances in the knowledge of EMT as it occurs in breast development and carcinoma and prostate cancer progression, and highlights the role that MET plays in cancer metastasis. Finally, perspectives from a clinical and translational viewpoint are discussed. J. Cell. Physiol. 213: 374–383, 2007.

Kallikreins on Steroids: Structure, Function, and Hormonal Regulation of Prostate-Specific Antigen and the Extended Kallikrein Locus

The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and putative proteolytic functions. The kallikrein family is also emerging as a rich source of disease biomarkers with KLK3, commonly known as prostate-specific antigen, being the current serum biomarker for prostate cancer. The kallikrein locus is also notable because it is extraordinarily responsive to steroids and other hormones. Indeed, at least 14 functional hormone response elements have been identified in the kallikrein locus. A more comprehensive understanding of the transcriptional regulation of kallikreins may help the field make more informed hypotheses about the physiological functions of kallikreins and their effectiveness as biomarkers. In this review, we describe the organization of the kallikrein locus and the structure of kallikrein genes and proteins. We also focus on the transcriptional regulation of kallikreins by androgens, progestins, glucocorticoids, mineralocorticoids, estrogens, and other hormones in animal models and human prostate, breast, and reproductive tract tissues. The interaction of the androgen receptor with androgen response elements in the promoter and enhancer of KLK2 and KLK3 is also summarized in detail. There is evidence that all kallikreins are regulated by multiple nuclear receptors. Yet, apart from KLK2 and KLK3, it is not clear whether all kallikreins are direct transcriptional targets. Therefore, we argue that gaining more detailed information about the mechanisms that regulate kallikrein expression should be a priority of future studies and that the kallikrein locus will continue to be an important model in the era of genome-wide analyses.

Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells.

Nodal is a member of the transforming growth factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity.

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 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.

The role of kallikrein-related peptidases in prostate cancer: potential involvement in an epithelial to mesenchymal transition.

Abstract Several members of the kallikrein-related peptidase family of serine proteases have proteolytic activities that may affect cancer progression; however, the in vivo significance of these activities remains uncertain. We have demonstrated that expression of PSA or KLK4, but not KLK2, in PC-3 prostate cancer cells changed the cellular morphology from epithelial to spindle-shaped, markedly reduced E-cadherin expression, increased vimentin expression and increased cellular migration. These changes are indicative of an epithelial to mesenchymal transition (EMT), a process important in embryonic development and cancer progression. The potential novel role of kallikrein-related peptidases in this process is the focus of this brief review.

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.

Germline BRCA2 mutations drive prostate cancers with distinct evolutionary trajectories

Germline mutations in the BRCA2 tumour suppressor are associated with both an increased lifetime risk of developing prostate cancer (PCa) and increased risk of aggressive disease. To understand this aggression, here we profile the genomes and methylomes of localized PCa from 14 carriers of deleterious germline BRCA2 mutations (BRCA2-mutant PCa). We show that BRCA2-mutant PCa harbour increased genomic instability and a mutational profile that more closely resembles metastastic than localized disease. BRCA2-mutant PCa shows genomic and epigenomic dysregulation of the MED12L/MED12 axis, which is frequently dysregulated in metastatic castration-resistant prostate cancer (mCRPC). This dysregulation is enriched in BRCA2-mutant PCa harbouring intraductal carcinoma (IDC). Microdissection and sequencing of IDC and juxtaposed adjacent non-IDC invasive carcinoma in 10 patients demonstrates a common ancestor to both histopathologies. Overall we show that localized castration-sensitive BRCA2-mutant tumours are uniquely aggressive, due to de novo aberration in genes usually associated with metastatic disease, justifying aggressive initial treatment.

Direct Progesterone Receptor and Indirect Androgen Receptor Interactions with the Kallikrein-Related Peptidase 4 Gene Promoter in Breast and Prostate Cancer

Kallikrein 4 (KLK4) is a member of the human KLK gene family of serine proteases, many of which are implicated in hormone-dependent cancers. Like other KLKs, such as KLK3/PSA and KLK2, KLK4 gene expression is also regulated by steroid hormones in hormone-dependent cancers, although the transcriptional mechanisms are ill defined. Here, we have investigated the mechanisms mediating the hormonal regulation of KLK4 in breast (T47D) and prostate (LNCaP and 22Rv1) cancer cells. We have shown that KLK4 is only expressed in breast and prostate cancers that express the progesterone receptor (PR) and androgen receptor (AR), respectively. Expression analysis in PR- and AR-positive cells showed that the two predominant KLK4 variants that use either TIS1 or TIS2a/b are both up-regulated by progesterone in T47D cells and androgens in LNCaP cells. Two putative hormone response elements, K4.pPRE and K4.pARE at −2419 bp and −1005 bp, respectively, were identified in silico. Electrophoretic mobility shift assays and luciferase reporter experiments suggest that neither K4.pARE nor ∼2.8 kb of the KLK4 promoter interacts directly with the AR to mediate KLK4 expression in LNCaP and 22Rv1 cells. However, we have shown that K4.pPRE interacts directly with the PR to up-regulate KLK4 gene expression in T47D cells. Further, chromatin immunoprecipitation experiments showed a time-dependent recruitment of the PR to the KLK4 promoter (−2496 to −2283), which harbors K4.pPRE. This is the first study to show that progesterone-regulated KLK4 expression in T47D cells is mediated partly by a hormone response element (K4.pPRE) at −2419 bp. (Mol Cancer Res 2009;7(1):129–41)

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.