Neuronal Trans-Differentiation in Prostate Cancer Cells.

BACKGROUND: Neuroendocrine (NE) differentiation in prostate cancer (PCa) is an aggressive phenotype associated with therapy resistance. The complete phenotype of these cells is poorly understood. Clinical classification is based predominantly on the expression of standard NE markers. METHODS: We analyzed the phenotype of NE carcinoma of the prostate utilizing in vitro methods, in silico, and immunohistochemical analyses of human disease. RESULTS: LNCaP cells, subjected to a variety of stressors (0.1% [v/v] fetal bovine serum, cyclic AMP) induced a reproducible phenotype consistent with neuronal trans-differentiation. Cells developed long cytoplasmic processes resembling neurons. As expected, serum deprived cells had decreased expression in androgen receptor and prostate specific antigen. A significant increase in neuronal markers also was observed. Gene array analysis demonstrated that LNCaP cells subjected to low serum or cAMP showed statistically significant manifestation of a human brain gene expression signature. In an in silico experiment using human data, we identified that only hormone resistant metastatic prostate cancer showed enrichment of the "brain profile." Gene ontology analysis demonstrated categories involved in neuronal differentiation. Three neuronal markers were validated in a large human tissue cohort. CONCLUSION: This study proposes that the later stages of PCa evolution involves neuronal trans-differentiation, which would enable PCa cells to acquire independence from the neural axis, critical in primary tumors. Prostate 76:1312-1325, 2016. © 2016 Wiley Periodicals, Inc.

Recruitment of CD34(+) fibroblasts in tumor-associated reactive stroma: the reactive microvasculature hypothesis.

Reactive stroma co-evolves with cancer, exhibiting tumor-promoting properties. It is also evident at sites of wound repair and fibrosis, playing a key role in tissue homeostasis. The specific cell types of origin and the spatial/temporal patterns of reactive stroma initiation are poorly understood. In this study, we evaluated human tumor tissue arrays by using multiple labeled, quantitative, spectral deconvolution microscopy. We report here a novel CD34/vimentin dual-positive reactive fibroblast that is observed in the cancer microenvironment of human breast, colon, lung, pancreas, thyroid, prostate, and astrocytoma. Recruitment of these cells occurred in xenograft tumors and Matrigel plugs in vivo and was also observed in stromal nodules associated with human benign prostatic hyperplasia. Because spatial and temporal data suggested the microvasculature as a common site of origin for these cells, we analyzed microvasculature fragments in organ culture. Interestingly, fibroblasts with identical phenotypic properties and markers expanded radially from microvasculature explants. We propose the concept of reactive microvasculature for the evolution of reactive stroma at sites of epithelial disruption common in both benign and malignant disorders. Data suggest that the reactive stroma response is conserved among tissues, in normal repair, and in different human cancers. A more clear understanding of the nature and origin of reactive stroma is needed to identify novel therapeutic targets in cancer and fibrosis.

Inducible FGFR-1 activation leads to irreversible prostate adenocarcinoma and an epithelial-to-mesenchymal transition.

Fibroblast Growth Factor Receptor-1 (FGFR1) is commonly overexpressed in advanced prostate cancer (PCa). To investigate causality, we utilized an inducible FGFR1 (iFGFR1) prostate mouse model. Activation of iFGFR1 with chemical inducers of dimerization (CID) led to highly synchronous, step-wise progression to adenocarcinoma that is linked to an epithelial-to-mesenchymal transition (EMT). iFGFR1 inactivation by CID withdrawal led to full reversion of prostatic intraepithelial neoplasia, whereas PCa lesions became iFGFR1-independent. Gene expression profiling at distinct stages of tumor progression revealed an increase in EMT-associated Sox9 and changes in the Wnt signaling pathway, including Fzd4, which was validated in human PCa. The iFGFR1 model clearly implicates FGFR1 in PCa progression and demonstrates how CID-inducible models can help evaluate candidate molecules in tumor progression and maintenance.

Determining prostate cancer-specific death through quantification of stromogenic carcinoma area in prostatectomy specimens.

We previously reported that reactive stroma grading in prostate cancer (PCa) is predictive of biochemical recurrence in prostatectomies and biopsies. In this study, we tested whether quantifying the percentage of reactive stromal grade 3 (RSG 3; stromogenic carcinoma pattern) in the entire tumor is predictive of PCa-specific death. Whole-mount prostatectomies operated by a single surgeon obtained between 1983 and 1998 were reviewed. Reactive stroma was evaluated as described previously, and areas of RSG 3 in the entire tumor were registered as percentages of total tumor. Statistical analysis was performed using Spearman, Kaplan-Meier, and Cox analyses. In all, 872 cases were evaluable. Quantification of RSG 3 percentage was an independent predictor of biochemical recurrence, analyzed as a continuous or grouped variable. Patients with higher RSG 3 percentages (larger tumor areas with RSG 3) had a significantly decreased biochemical recurrence-free survival than those with a lower RSG 3 percentage, even within the Gleason score 7 subset of patients. A nomogram introduced this new variable to the model. Furthermore, quantification of RSG 3 percentage was significantly predictive of PCa-specific death. Quantification of the RSG 3 (stromogenic carcinoma) area in PCa provides additional novel information on prognosis. These data substantiate the concept that the tumor microenvironment holds significant predictive information, as well as biological significance.

Neurogenesis in colorectal cancer is a marker of aggressive tumor behavior and poor outcomes.

BACKGROUND: Colorectal cancer staging criteria do not rely on examination of neuronal tissue. The authors previously demonstrated that perineural invasion is an independent prognostic factor of outcomes in colorectal cancer. For the current study, they hypothesized that neurogenesis occurs in colorectal cancer and portends an aggressive tumor phenotype. METHODS: In total, samples from 236 patients with colorectal cancer were used to create a tissue array and database. Tissue array slides were immunostained for protein gene product 9.5 (PGP9.5) to identify nerve tissue. The correlation between markers of neurogenesis and oncologic outcomes was determined. The effect of colorectal cancer cells on stimulating neurogenesis in vitro was evaluated using a dorsal root ganglia coculture model. RESULTS: Patients whose tumors exhibited high degrees of neurogenesis had 50% reductions in 5-year overall survival and disease-free survival compared with patients whose tumors contained no detectable neurogenesis (P = .002 and P = .006, respectively). Patients with stage II disease and high degrees of neurogenesis had greater reductions in 5-year overall survival and disease-free survival compared with lymph node-negative patients with no neurogenesis (P = .002 and P = .008, respectively). Patients with stage II disease and high degrees of neurogenesis had lower 5-year overall survival and disease-free survival compared with patients who had stage III disease with no neurogenesis (P = .01 and P = .008, respectively). Colorectal cancer cells stimulated neurogenesis and exhibited evidence of neuroepithelial interactions between nerves and tumor cells in vitro. CONCLUSIONS: Neurogenesis in colorectal cancer appeared to play a critical role in colorectal cancer progression. Furthermore, the current results indicated that neurogenesis functions as an independent predictor of outcomes and may play a role in therapy stratification for patients with lymph node-negative disease.

Detection of xenotropic murine leukemia virus-related virus in normal and tumor tissue of patients from the southern United States with prostate cancer is dependent on specific polymerase chain reaction conditions.

BACKGROUND: There are questions regarding the prevalence of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and its association with the RNASEL R462Q polymorphism. We therefore investigated whether XMRV infection could be found in patients with prostate cancer from the southern United States, and we sought to verify the association with the R462Q. METHODS: Prostate tissue specimens of 144 patients with prostate cancer from the southern United States were genotyped for R462Q by real time polymerase chain reaction allelic discrimination and were screened for XMRV proviral DNA by nested polymerase chain reaction specific for the env gene. RESULTS: The R462Q polymorphism was found at an allelic frequency of 0.33. XMRV was detected in 32 (22%) of the 144 patients. Patients were significantly more likely to test positive for XMRV in both tumor and normal tissue rather than either alone (κ = 0.64). A positive result for XMRV was not significantly correlated with the R462Q polymorphism (P = .82) or clinical pathological parameters of prostate cancer, including Gleason score (P = .29). CONCLUSIONS: XMRV is detectable in normal and tumor prostate tissue from patients with prostate cancer, independent of R462Q. The presence of XMRV in normal tissue suggests that infection may precede cancer onset.

Multiphoton Microscopy for Identifying Collagen Signatures Associated with Biochemical Recurrence in Prostate Cancer Patients.

Prostate cancer is a heterogeneous disease that remains dormant for long periods or acts aggressively with poor clinical outcomes. Identifying aggressive prostate tumor behavior using current glandular-focused histopathological criteria is challenging. Recent evidence has implicated the stroma in modulating prostate tumor behavior and in predicting post-surgical outcomes. However, the emergence of stromal signatures has been limited, due in part to the lack of adoption of imaging modalities for stromal-specific profiling. Herein, label-free multiphoton microscopy (MPM), with its ability to image tissue with stromal-specific contrast, is used to identify prostate stromal features associated with aggressive tumor behavior and clinical outcome. MPM was performed on unstained prostatectomy specimens from 59 patients and on biopsy specimens from 17 patients with known post-surgery recurrence status. MPM-identified collagen content, organization, and morphological tumor signatures were extracted for each patient and screened for association with recurrent disease. Compared to tumors from patients whose disease did not recur, tumors from patients with recurrent disease exhibited higher MPM-identified collagen amount and collagen fiber intensity signal and width. Our study shows an association between MPM-identified stromal collagen features of prostate tumors and post-surgical disease recurrence, suggesting their potential for prostate cancer risk assessment.

Improved prediction of prostate cancer recurrence through systems pathology.

We have developed an integrated, multidisciplinary methodology, termed systems pathology, to generate highly accurate predictive tools for complex diseases, using prostate cancer for the prototype. To predict the recurrence of prostate cancer following radical prostatectomy, defined by rising serum prostate-specific antigen (PSA), we used machine learning to develop a model based on clinicopathologic variables, histologic tumor characteristics, and cell type-specific quantification of biomarkers. The initial study was based on a cohort of 323 patients and identified that high levels of the androgen receptor, as detected by immunohistochemistry, were associated with a reduced time to PSA recurrence. The model predicted recurrence with high accuracy, as indicated by a concordance index in the validation set of 0.82, sensitivity of 96%, and specificity of 72%. We extended this approach, employing quantitative multiplex immunofluorescence, on an expanded cohort of 682 patients. The model again predicted PSA recurrence with high accuracy, concordance index being 0.77, sensitivity of 77% and specificity of 72%. The androgen receptor was selected, along with 5 clinicopathologic features (seminal vesicle invasion, biopsy Gleason score, extracapsular extension, preoperative PSA, and dominant prostatectomy Gleason grade) as well as 2 histologic features (texture of epithelial nuclei and cytoplasm in tumor only regions). This robust platform has broad applications in patient diagnosis, treatment management, and prognostication.

Prognostic value of Akt-1 in human prostate cancer: a computerized quantitative assessment with quantum dot technology.

BACKGROUND: Akt/protein kinase B signaling pathway has been implicated in tumorigenesis and progression. Previous studies showed the predictive potential of p-Akt-1, but total Akt-1 could provide more reliable information. We used image deconvolution, nanotechnology (quantum dots), and image analysis to improve Akt-1 quantification. DESIGN: This tissue microarray study included 840 radical prostatectomy cases. Slides were incubated with primary antibody against nonphosphorylated Akt-1 (Akt-1) followed by biotinylated secondary antibody and then by Qdot655 streptavidin conjugate. Slides were imaged under fluorescence microscopy and spectral deconvolution (Nuance) and quantified using plug-in image analysis software. Average intensity of Akt-1 signal was measured and subject to statistical analysis. Multivariate analysis (Cox regression) was applied to assess the prognostic value of Akt-1 for biochemical recurrence and prostate cancer-specific death. Akt-1 expression was also examined for correlations with Ki-67 index and apoptotic index in our database. RESULT: Akt-1 was inversely correlated with apoptotic index (rho = -0.203; P = 0.004) but not with Ki-67 index. The correlation between Akt and p-Akt is significant but weak (P = 0.0496; R(2) = 0.118). On multivariate analysis Akt-1 was independently predictive of biochemical recurrence [hazard ratio, 2.863 (95% confidence interval, 1.127-7.271); P = 0.0270]. Akt-1 level is also predictive of prostate cancer-specific death (P = 0.0376). CONCLUSION: High levels of Akt-1, assessed by quantum dots, deconvolution imaging, and image analysis, are associated with a higher risk of biochemical recurrence and prostate cancer-specific death.

Cytoplasmic accumulation of glycogen synthase kinase-3beta is associated with aggressive clinicopathological features in human prostate cancer.

BACKGROUND: Activation of glycogen synthase kinase-3 (GSK-3) is involved in the regulation of cell growth, differentiation, mobility, proliferation and survival. However, its clinicopathologic significance remains unclear in prostate cancer (PCa). MATERIALS AND METHODS: A tissue microarray was produced from 640 samples. Sections were immunostained with an antibody against the non-phosphorylated form of GSK-3(GSK-3beta) and were digitized. Spearman correlation test was processed for correlations between GSK-3beta and biological and clinicopathological variables. The prognostic value of GSK-3beta was analyzed by Cox Regression model. RESULTS: Cytoplasmic GSK-3beta was higher in PCa than in normal prostate (mean expression index 4.55 vs. 3.50, p<0.0001). Conversely, nuclear expression was higher in normal prostate than that in PCa (3.38 vs. 2.04, p<0.0001). Cytoplasmic levels of GSK-3beta were correlated with clinical stage (rho=0.095, p=0.0337), lymph node metastasis (rho=0.116, p=0.0096), extracapsular extension (rho=0.092, p=0.0392), and Gleason score (rho=0.167, p=0.0002). Increased cytoplasmic GSK-3beta expression was correlated with high Ki-67 labeling index (rho=0.319, p<0.0001), low apoptotic index by TUNEL (rho=-0.118, p=0.0134), high levels of androgen receptor (rho=0.292, p<0.0001) and p-Akt (rho=0.396, p<0.0001). Patients with higher cytoplasmic levels of GSK-3beta had a two-fold risk of biochemical recurrence-free survival compared to those with lower levels of GSK-3beta [HR 1.934 (1.020-3.667), p=0.043]. CONCLUSION: Cytoplasmic accumulation of GSK-3beta is potentially associated with a pro-survival mechanism that promotes PCa development and progression.

Cancer-related axonogenesis and neurogenesis in prostate cancer.

PURPOSE: Perineural invasion is the only interaction between cancer cells and nerves studied to date. It is a symbiotic relationship between cancer and nerves that results in growth advantage for both. In this article, we present data on a novel biological phenomenon, cancer-related axonogenesis and neurogenesis. EXPERIMENTAL DESIGN: We identify spatial and temporal associations between increased nerve density and preneoplastic and neoplastic lesions of the human prostate. RESULTS: Nerve density was increased in cancer areas as well as in preneoplastic lesions compared with controls. Two- and three-dimensional reconstructions of entire prostates confirmed axonogenesis in human tumors. Furthermore, patients with prostate cancer had increased numbers of neurons in their prostatic ganglia compared with controls, corroborating neurogenesis. Finally, two in vitro models confirmed that cancer cells, particularly when interacting with nerves in perineural invasion, induce neurite outgrowth in prostate cancer. Neurogenesis is correlated with features of aggressive prostate cancer and with recurrence in prostate cancer. We also present a putative regulatory mechanism based on semaphorin 4F (S4F). S4F is overexpressed in cancers cells in the perineural in vitro model. Overexpression of S4F in prostate cancer cells induces neurogenesis in the N1E-115 neurogenesis assay and S4F inhibition by small interfering RNA blocks this effect. CONCLUSIONS: This is the first description of cancer-related neurogenesis and its putative regulatory mechanism.

Intraosseous malignant peripheral nerve sheath tumor of the sacrum in a patient with neurofibromatosis type I.

Malignant intraosseous peripheral nerve sheath tumor is a very rare malignancy most commonly seen in patients with neurofibromatosis type 1. This tumor almost exclusively occurs in the maxillofacial region, with manifestation of this tumor in other regions of the skeleton infrequently reported. We describe a 23-year-old female with previously undiagnosed neurofibromatosis type 1 presenting with lower extremity weakness, paresthesias, and bowel/bladder symptoms. The patient had an aneurysmal lytic bone lesion centered in the upper sacrum with invasion of the L5 vertebral body. On MRI, the lesion was homogeneously isointense to muscle on T1, heterogeneously hyperintense to muscle on T2, and demonstrated homogeneously avid contrast enhancement. Multiple additional small lesions with similar imaging characteristics were identified in the paraspinal soft tissues. Low grade malignant peripheral nerve sheath tumor of the sacrum was diagnosed on biopsy. The patient was treated with sacral resection and radiation therapy for local disease control.

Moving Beyond Gleason Scoring.

CONTEXT.­: The combination of grading and staging is the basis of current standard of care for prediction for most cancers. D. F. Gleason created the current prostate cancer (PCa) grading system. This system has been modified several times. Molecular data have been added. Currently, all grading systems are cancer-cell based. OBJECTIVE.­: To review the literature available on host response measures as reactive stroma grading and stromogenic carcinoma, and their predictive ability for PCa biochemical recurrence and PCa-specific death. DATA SOURCES.­: Our own experience has shown that reactive stroma grading and the subsequently binarized system (stromogenic carcinoma) can independently predict biochemical recurrence and/or PCa-specific death, particularly in patients with a Gleason score of 6 or 7. Stromogenic carcinoma has been validated by 4 other independent groups in at least 3 continents. CONCLUSIONS.­: Broders grading and Dukes staging have been combined to form the most powerful prognostic tools in standard of care. The time has come for us to incorporate measures of host response (stromogenic carcinoma) into the arsenal of elements we use to predict cancer survival, without abandoning what we know works. These data also suggest that our current definition of PCa might need some revision.

Reprint of: Stromogenic prostatic carcinoma pattern (carcinomas with reactive stromal grade 3) in needle biopsies predicts biochemical recurrence-free survival in patients after radical prostatectomy.

We previously reported that reactive stromal grading in radical prostatectomies is a predictor of recurrence and that reactive stromal grading 0 and 3 are associated with lower biochemical recurrence-free survival rates than reactive stromal grading 1 and 2. We explored the prognostic significance of reactive stromal grading in preoperative needle biopsies. At Baylor College of Medicine, 224 cases of prostatic carcinoma were diagnosed by needle biopsy. Reactive stromal grading was evaluated on hematoxylin-eosin (H&E)-stained sections on the basis of previously described criteria: grade 0, with 0% to 5% reactive stroma; grade 1, 6% to 15%; grade 2, 16% to 50%; grade 3, 51% to 100%, or at least a 1:1 ratio between glands and stroma. Kaplan-Meier and Cox proportional hazard analyses were used. Reactive stromal grading distribution was as follows: reactive stromal grading 0, 1 case (0.5%); reactive stromal grading 1, 149 cases (66.5%); reactive stromal grading 2, 59 cases (26.3%); reactive stromal grading 3, 15 cases (6.7%). Reactive stromal grading in biopsies was correlated with adverse clinicopathologic parameters in the prostatectomy. Patients with reactive stromal grading 1 and 2 had better survival than those with 0 and 3 (P = .0034). Reactive stromal grading was an independent predictor of recurrence (hazard ratio = 1.953; P = .0174). Reactive stromal grading is independent of Gleason 4 + 3 and 3 + 4 in patients with a Gleason score of 7. Quantitation of reactive stroma and recognition of the stromogenic carcinoma in H&E-stained biopsies is useful to predict biochemical recurrence in prostate carcinoma patients independent of Gleason grade and prostate-specific antigen.

Malignant advanced granulosa cell tumor of the adult testis: case report and review of the literature.

Testicular granulosa cell tumors (GCTs) are very rare neoplasms. Although adult GCTs are thought to have a relatively indolent course, several reports have demonstrated the malignant potential of these lesions. In case of distant metastases, the overall survival is very short. To date, there is no well-established treatment for these tumors owing to poor results and very rapid progression. A 55-year-old male patient was diagnosed with a testicular GCT with distant lung metastases. He underwent surgical treatment with orchiectomy and adjuvant polychemotherapy (cisplantine, etoposide, and bleomycine) as well as metastasectomy of the right lung. We report the first case of a successfully treated testicular GCT with bipulmonary metastases at initial diagnosis. Thirty-nine months after treatment, the patient is alive with no evidence of disease. We subsequently reviewed all reported cases of an adult GCT in the published literature (25 published cases). This review will summarize all reported cases and discuss treatment options. The current case suggests that a combination of varying treatment modalities could be a promising and reasonable way to manage malignant advanced GCT of the adult testis.

Androgens modulate expression of transcription intermediary factor 2, an androgen receptor coactivator whose expression level correlates with early biochemical recurrence in prostate cancer.

Prostate cancer is an androgen-dependent disease; metastatic prostate cancer is typically treated by androgen receptor (AR) blockade. Recurrence after androgen ablation and evidence that AR continues to play a role in many prostate cancers has led to an examination of other factors that potentiate AR activity. AR is a ligand-activated transcription factor whose activity is regulated not only by hormone but also by the levels of coactivators recruited by AR to facilitate transcription. We sought to assess the consequences of reducing expression of the transcription intermediary factor 2 (TIF2) coactivator on prostate cancer cell growth and AR action in cell lines to examine TIF2 expression in prostate cancer and to correlate expression with clinical outcome. Depletion of TIF2 reduced expression of AR-induced target genes and slowed proliferation of AR-dependent and AR-independent prostate cancer cells. Remarkably, we found that TIF2 expression is directly repressed by high levels of androgens in multiple AR-expressing cell lines. Expression of a reporter containing 5'-flanking region of the TIF2 was repressed both by androgens and by the antagonist, Casodex. Expression of TIF2 correlates with biochemical (prostate-specific antigen) recurrence (P = 0.0136). In agreement with our in vitro findings, the highest expression of TIF2 was found in patients whose cancer relapsed after androgen ablation therapy, supporting the idea that AR blockade might activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium. The elevated expression of TIF2 at low hormone levels likely aids in inducing AR activity under these conditions; treatment with Casodex has the potential to counteract this induction.

Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.

Caveolin-1 (cav-1) is a major scaffolding component of cell membrane invaginations (caveolae). It is involved in sequestering numerous effectors and signaling molecules and has antiapototic activities in prostate cancer. Perineural invasion (PNI) is associated with decreased apoptosis of cancer cells both in human tissues and the in vitro PNI model. We show here that stromal (perineurium) production of cav-1 is involved in a paracrine antiapoptotic loop in PNI. Transforming growth factor-beta1 is up-regulated in the cancer cells as they approach the nerve and is thought to up-regulate cav-1 in the perineurium of nerves with prostate cancer. Cav-1 is then secreted into the microenvironment and used by prostate cancer cells to inhibit apoptosis. In the in vitro PNI model, this phenomenon is partially reversed by neutralizing cav-1 antibodies or using ganglia from cav-1 knockout mice. Our results show a novel paracrine mechanism used by the prostate cancer in PNI to increase their proliferative activity and decrease apoptosis.

Forkhead protein FKHR and its phosphorylated form p-FKHR in human prostate cancer.

In vitro studies suggest that the proapoptotic function of forkhead protein FKHR is probably inactivated by means of phosphorylation through the protein kinase B pathway. However, the clinical significance of FKHR in prostate cancer remains unclear. Six hundred forty radical prostatectomies were used for building tissue microarrays. Slides were stained with antibodies against FKHR and phosphorylated FKHR (p-FKHR). Correlations with clinicopathologic parameters were analyzed by Spearman rank test. Cox regression test and Kaplan-Meier test were used to determine the probability of disease recurrence, which is defined as a serum prostate-specific antigen (PSA) level greater than 0.4 ng/mL after radical prostatectomy. Nuclear FKHR level was higher in normal prostate than in benign prostatic hyperplasia and prostate cancer (P = .0000). Nuclear expression of FKHR was correlated with preoperative PSA level (rho = 0.108, P = .029), extracapsular extension (rho = 0.137, P = .005), and seminal vesicle invasion (rho = 0.101, P = .039). FKHR expression was not a significant indicator of biochemical failure by either univariate or multivariate analysis. Nuclear p-FKHR expression correlated with patients' age (rho = 0.179, P = .0003), Gleason score (rho = 0.130, P = .0083), extracapsular extension (rho = 0.227, P = .0000), clinical stage (Union Internationale Contre le Cancer system) (rho = 0.166, P = .0007), and lymph node status (rho = 0.101, P = .0401). Cytoplasmic p-FKHR correlated with patients' age (rho = 0.146, P = .0030) and clinical stage (rho = 0.117, P = .0180). Cytoplasmic p-FKHR was a significant indicator of biochemical recurrence (P = .0164; hazard ratio, 1.114-2.929). Nuclear p-FKHR strongly correlated with phosphorylated protein kinase B (rho = 0.368, P = .0000), androgen receptor (rho = 0.385, P = .0000), and Skp-2 (rho = 0.170, P = .0036). Our data suggest that the proapoptotic role of FKHR is probably regulated by several signaling pathways in prostate cancer.

Enhanced survival in perineural invasion of pancreatic cancer: an in vitro approach.

Pancreatic cancer (PanCa) is characterized by perineural invasion (PNI), early lymph node and liver metastasis, and poor prognosis. PNI is one of the important causes of local recurrence. Little is known about the mechanism of PNI in PanCa. We presented a novel model system that may shed light on the mystery of PNI in PanCa. In this study, mouse dorsal root ganglia (DRGs) and human PanCa cell line (MIA PaCa-2) were cocultured in Matrigel matrix (BD Biosciences, San Jose, CA) to build this PNI model. MIA PaCa-2 cell line alone (control 1) or DRG alone (control 2) was cultured with Matrigel matrix as controls. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were observed under inverted microscopy and then were photographed and quantitated with the Optimas imaging system (Optimas Corp., Bothell, MA). At day 14, both the experimental and control 2 samples were harvested and subjected to total RNA isolation and fixed in paraffin-embedded blocks. Slides cut from paraffin blocks were studied with Ki-67 immunostaining and TUNEL assay. Gene profiling was performed using complementary DNA microarray. Overexpressed target genes were verified by quantitative reverse transcriptase polymerase chain reaction. The results showed that reciprocity was observed between neurites and MIA PaCa colonies with 24 hours of coculture. Neurite outgrowth was stimulated in the presence of pancreatic carcinoma cells, which showed 2-fold more area than did control 2. After 72 hours, MIA PaCa colonies cocultured with DRG exhibited 58% more colony area than did control 1. The Ki-67 index of the DRG/MIA PaCa cells (mean, 5.02%) was significantly higher than that in control 1 (mean, 1.18%) (P < .05); in contrast, the apoptotic index in the DRG/MIA PaCa cells was significantly lower (mean, 0.45%) than that in the control 1 (mean, 1.85%) (P < .001). Prosurvival genes MALT1 and TRAF were increased 2-fold in DRG/MIA PaCa compared with controls. We demonstrated that neural-epithelial interaction is a mutually beneficial process for the growth of nerves and PanCa cells. It is possible that oncogenes and growth factors might act synergistically in promoting proliferation and/or inhibiting apoptosis, a survival strategy crucial to the development of PNI in PanCa.