Beatrice Knudsen

The Gene Expression Program of Prostate Fibroblast Senescence Modulates Neoplastic Epithelial Cell Proliferation through Paracrine Mechanisms

The greatest risk factor for developing carcinoma of the prostate is advanced age. Potential molecular and physiologic contributors to the frequency of cancer occurrence in older individuals include the accumulation of somatic mutations through defects in genome maintenance, epigenetic gene silencing, oxidative stress, loss of immune surveillance, telomere dysfunction, chronic inflammation, and alterations in tissue microenvironment. In this context, the process of prostate carcinogenesis can be influenced through interactions between intrinsic cellular alterations and the extrinsic microenvironment and macroenvironment, both of which change substantially as a consequence of aging. In this study, we sought to characterize the molecular alterations that occur during the process of prostate fibroblast senescence to identify factors in the aged tissue microenvironment capable of promoting the proliferation and potentially the neoplastic progression of prostate epithelium. We evaluated three mechanisms leading to cell senescence: oxidative stress, DNA damage, and replicative exhaustion. We identified a consistent program of gene expression that includes a subset of paracrine factors capable of influencing adjacent prostate epithelial growth. Both direct coculture and conditioned medium from senescent prostate fibroblasts stimulated epithelial cell proliferation, 3-fold and 2-fold, respectively. The paracrine-acting proteins fibroblast growth factor 7, hepatocyte growth factor, and amphiregulin (AREG) were elevated in the extracellular environment of senescent prostate fibroblasts. Exogenous AREG alone stimulated prostate epithelial cell growth, and neutralizing antibodies and small interfering RNA targeting AREG attenuated, but did not completely abrogate the growth-promoting effects of senescent fibroblast conditioned medium. These results support the concept that aging-related changes in the prostate microenvironment may contribute to the progression of prostate neoplasia.

Oral Cavity and Esophageal Carcinogenesis Modeled in Carcinogen-Treated Mice

Purpose: Squamous cell carcinoma of the oral cavity is one of the most common human neoplasms, and prevention of these carcinomas requires a better understanding of the carcinogenesis process and a model system in which cancer chemoprevention agents can be tested. We have developed a mouse model using the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in the drinking water to induce tumorigenesis in the mouse oral cavity. Experimental Design: 4-NQO was delivered by tongue painting or drinking water to two mouse strains, CBA and C57Bl/6. The incidences of oral cavity carcinogenesis were then compared. In addition, we examined the expression of some of the molecular markers associated with the process of human oral cavity and esophageal carcinogenesis, such as keratin (K) 1, K14, p16, and epidermal growth factor receptor, by immunohistochemistry. Results: After treatment with 4-NQO in the drinking water, massive tumors were observed on the tongues of both CBA and C57Bl/6 female mice. Pathological analyses indicated that flat squamous dysplasias, exophytic papillary squamous tumors (papillomas), and invasive squamous cell carcinomas were present. Immunohistochemistry analyses showed that 4-NQO changed the expression patterns of the intermediate filament proteins K14 and K1. K14 was expressed in the epithelial suprabasal layers, in addition to the basal layer, in tongues from carcinogen-treated animals. In contrast, control animals expressed K14 only in the basal layer. Moreover, we observed more bromodeoxyuridine staining in the tongue epithelia of 4-NQO-treated mice. Reduced expression of the cell cycle inhibitor, p16, was observed, whereas 4-NQO treatment caused an increase in epidermal growth factor receptor expression in the mouse tongues. Interestingly, similar features of carcinogenesis, including multiple, large (up to 0.5 cm) exophytic papillary squamous tumors and invasive squamous cell carcinomas, increased bromodeoxyuridine staining, and increased K14 expression, were also observed in the esophagi of 4-NQO-treated mice. However, no tumors were observed in the remainder of digestive tract (including the forestomach, intestine, and colon) or in the lungs or livers of 4-NQO-treated mice. These results indicate that this murine 4-NQO-induced oral and esophageal carcinogenesis model simulates many aspects of human oral cavity and esophageal carcinogenesis. Conclusions: The availability of this mouse model should permit analysis of oral cavity and esophageal cancer development in various mutant and transgenic mouse strains. This model will also allow testing of cancer chemopreventive drugs in various transgenic mouse strains.

Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells

ABSTRACT Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cγ, and activation of the Ras/Erk and phosphatidylinositol 3′-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G1 cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G1 cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.

Influence of Surgical Manipulation on Prostate Gene Expression: Implications for Molecular Correlates of Treatment Effects and Disease Prognosis

PURPOSE Measurements of tissue gene expression are increasingly used for disease stratification, clinical trial eligibility, and assessment of neoadjuvant therapy response. However, the method of tissue acquisition alone could significantly influence the expression of specific transcripts or proteins. This study examines whether there are transcript alterations associated with surgical resection of the prostate gland by radical retropubic prostatectomy. MATERIALS AND METHODS Twelve patients with clinically localized prostate cancer underwent immediate in situ prostate biopsy after induction of anesthesia for radical prostatectomy. Ex vivo prostate biopsies were performed immediately after surgical removal. Prostate epithelium was acquired by laser-capture microdissection, and transcript abundance levels were quantitated by cDNA microarray hybridization and confirmed by quantitative polymerase chain reaction. Data were analyzed by paired, two-sample t test using Statistical Analysis of Microarray algorithms, and linear models were fit as a function of clinical characteristics. RESULTS Of 5,753 cDNAs with measurable expression in prostate epithelium, 88 (1.5%) were altered as a result of surgery (false-discovery rate < or = 10%), representing 62 unique genes. These included transcripts encoding acute phase response proteins, IER2 and JUNB, and regulators of cell proliferation, p21Cip1 and KLF6. Of the clinical characteristics examined, including patient age, prostate volume, serum prostate-specific antigen, blood loss, and operative time, only gland volume was significantly and negatively associated with the magnitude of gene expression difference between pre- and postsurgical specimens. CONCLUSION Surgical manipulation results in significant gene expression changes. Molecular analyses of surgical samples should recognize that transcript alterations occur rapidly, and these results are important when designing and analyzing molecular correlates of clinical studies.

Circulating microRNA Profiling Identifies a Subset of Metastatic Prostate Cancer Patients with Evidence of Cancer-Associated Hypoxia

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.

The critical role of the pathologist in determining eligibility for active surveillance as a management option in patients with prostate cancer: consensus statement with recommendations supported by the College of American Pathologists, International Society of Urological Pathology, Association of Directors of Anatomic and Surgical Pathology, the New Zealand Society of Pathologists, and the Prostate Cancer Foundation.

CONTEXT Prostate cancer remains a significant public health problem. Recent publications of randomized trials and the US Preventive Services Task Force recommendations have drawn attention to overtreatment of localized, low-risk prostate cancer. Active surveillance, in which patients undergo regular visits with serum prostate-specific antigen tests and repeat prostate biopsies, rather than aggressive treatment with curative intent, may address overtreatment of low-risk prostate cancer. It is apparent that a greater awareness of the critical role of pathologists in determining eligibility for active surveillance is needed. OBJECTIVES To review the state of current knowledge about the role of active surveillance in the management of prostate cancer and to provide a multidisciplinary report focusing on pathologic parameters important to the successful identification of patients likely to succeed with active surveillance, to determine the role of molecular tests in increasing the safety of active surveillance, and to provide future directions. DESIGN Systematic review of literature on active surveillance for low-risk prostate cancer, pathologic parameters important for appropriate stratification, and issues regarding interobserver reproducibility. Expert panels were created to delineate the fundamental questions confronting the clinical and pathologic aspects of management of men on active surveillance. RESULTS Expert panelists identified pathologic parameters important for management and the related diagnostic and reporting issues. Consensus recommendations were generated where appropriate. CONCLUSIONS Active surveillance is an important management option for men with low-risk prostate cancer. Vital to this process is the critical role pathologic parameters have in identifying appropriate candidates for active surveillance. These findings need to be reproducible and consistently reported by surgical pathologists with accurate pathology reporting.

E-cadherin-mediated survival of androgen-receptor-expressing secretory prostate epithelial cells derived from a stratified in vitro differentiation model

The androgen receptor (AR) is expressed in differentiated secretory prostate epithelial cells in vivo. However, in the human prostate, it is unclear whether androgens directly promote the survival of secretory cells, or whether secretory cells survive through androgen-dependent signals from the prostate stroma. Biochemical and mechanistic studies have been hampered by inadequate cell-culture models. In particular, large-scale differentiation of prostate epithelial cells in culture has been difficult to achieve. Here, we describe the development of a differentiation system that is amenable to functional and biochemical analysis and its application to deciphering the survival pathways in differentiated AR-expressing epithelial cells. Confluent prostate epithelial cell cultures were treated with keratinocyte growth factor (KGF) and dihydrotestosterone. After 2 weeks, a suprabasal cell layer was formed in which cells no longer expressed α2, α3, α6, αv, β1 or β4 integrins or p63, K5, K14, EGFR, FGFR2IIIb or Bcl-2, but instead expressed AR and androgen-induced differentiation markers, including K18, K19, TMPRSS2, Nkx3.1, PMSA, KLK2 and secreted prostate-specific antigen (PSA). Differentiated prostate cell survival depended on E-cadherin and PI3K, but not KGF, androgen, AR or MAPK. Thus survival of differentiated prostate epithelial cells is mediated by cell-cell adhesion, and not through androgen activity or prostate stroma-derived KGF.

Low-Fat, Low-Glycemic Load Diet and Gene Expression in Human Prostate Epithelium: A Feasibility Study of Using cDNA Microarrays to Assess the Response to Dietary Intervention in Target Tissues

Purpose: We examined the feasibility of using gene expression changes in human prostate epithelium as a measure of response to a dietary intervention. Materials and Methods: Eight men with newly diagnosed prostate cancer were randomized to a low-fat/low-glycemic load intervention arm (<20% energy from fat and total daily glycemic load <100) or a “standard American” control arm (≈35% energy from fat and total daily glycemic load >200). Prostate tissue was collected before randomization and ∼6 weeks later, at the time of radical prostatectomy. Epithelium was acquired by laser capture microdissection, and transcript abundance levels were measured by cDNA microarray hybridization and confirmed by quantitative reverse transcription-PCR. Results: Men in the intervention arm consumed 39% less total energy (P = 0.004) and the difference in weight change between intervention and control arms was −6.1 kg (P = 0.02). In the intervention arm, 23 (0.46%) of 5,711 cDNAs with measurable expression were significantly altered (P < 0.05; false discovery rate, ≤10%). In the control arm, there were no significant changes in transcript expression, even when using a false discovery rate as high as 50%. Conclusions: A 6-week, low-fat/low-glycemic load diet was associated with significant gene expression changes in human prostate epithelium. These results show the feasibility of using prostate tissues collected at diagnosis and at surgery to study the effects of dietary manipulation on prostate tissue, which may give insight into the molecular mechanisms underlying the associations of diet and obesity with the development or progression of prostate cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2150–4)

Differential Gene Expression in Benign Prostate Epithelium of Men with and without Prostate Cancer: Evidence for a Prostate Cancer Field Effect

Background: Several malignancies are known to exhibit a “field effect,” whereby regions beyond tumor boundaries harbor histologic or molecular changes that are associated with cancer. We sought to determine if histologically benign prostate epithelium collected from men with prostate cancer exhibits features indicative of premalignancy or field effect. Experimental Design: Prostate needle biopsies from 15 men with high-grade (Gleason 8-10) prostate cancer and 15 age- and body mass index–matched controls were identified from a biospecimen repository. Benign epithelia from each patient were isolated by laser capture microdissection. RNA was isolated, amplified, and used for microarray hybridization. Quantitative PCR was used to determine the expression of specific genes of interest. Alterations in protein expression were analyzed through immunohistochemistry. Results: Overall patterns of gene expression in microdissected benign prostate-associated benign epithelium (BABE) and cancer-associated benign epithelium (CABE) were similar. Two genes previously associated with prostate cancer, PSMA and SSTR1, were significantly upregulated in the CABE group (false discovery rate <1%). Expression of other prostate cancer–associated genes, including ERG, HOXC4, HOXC5, and MME, were also increased in CABE by quantitative reverse transcription-PCR, although other genes commonly altered in prostate cancer were not different between the BABE and CABE samples. The expression of MME and PSMA proteins on immunohistochemistry coincided with their mRNA alterations. Conclusion: Gene expression profiles between benign epithelia of patients with and without prostate cancer are very similar. However, these tissues exhibit differences in the expression levels of several genes previously associated with prostate cancer development or progression. These differences may comprise a field effect and represent early events in carcinogenesis. Clin Cancer Res; 16(22); 5414–23. ©2010 AACR.

Consensus statement with recommendations on active surveillance inclusion criteria and definition of progression in men with localized prostate cancer: the critical role of the pathologist

Active surveillance (AS) is an important management option for men with low-risk, clinically localized prostate cancer. The clinical parameters for patient selection and definition of progression for AS protocols are evolving as data from several large cohorts matures. Vital to this process is the critical role pathologic parameters play in identifying appropriate candidates for AS. These findings need to be reproducible and consistently reported by surgical pathologists. This report highlights the importance of accurate pathology reporting as a critical component of these protocols.