Characterization of RNA-Binding Motif 3 (RBM3) Protein Levels and Nuclear Architecture Changes in Aggressive and Recurrent Prostate Cancer.

Background: The RNA-binding motif protein 3 (RBM3) has been shown to be up-regulated in several types of cancer, including prostate cancer (PCa), compared to normal tissues. Increased RBM3 nuclear expression has been linked to improved clinical outcomes. Aims: Given that RBM3 has been hypothesized to play a role in critical nuclear functions such as chromatin remodeling, DNA damage response, and other post-transcriptional processes, we sought to: (1) quantify RBM3 protein levels in archival PCa samples; (2) develop a nuclear morphometric model to determine if measures of RBM3 protein levels and nuclear features could be used to predict disease aggressiveness and biochemical recurrence. Methods & Results: This study utilized two tissue microarrays (TMAs) stained for RBM3 that included 80 total cases of PCa stratified by Gleason score. A software-mediated image processing algorithm identified RBM3-positive cancerous nuclei in the TMA samples and calculated twenty-two features quantifying RBM3 expression and nuclear architecture. Multivariate logistic regression (MLR) modeling was performed to determine if RBM3 levels and nuclear structural changes could predict PCa aggressiveness and biochemical recurrence (BCR). Leave-one-out cross validation (LOOCV) was used to provide insight on how the predictive capabilities of the feature set might behave with respect to an independent patient cohort to address issues such as model overfitting. RBM3 expression was found to be significantly downregulated in highly aggressive GS ≥ 8 PCa samples compared to other Gleason scores (P < 0.0001) and significantly down-regulated in recurrent PCa samples compared to non-recurrent samples (P = 0.0377). An eleven-feature nuclear morphometric MLR model accurately identified aggressive PCa, yielding a receiver operating characteristic area under the curve (ROC-AUC) of 0.90 (P < 0.0001) in the raw data set and 0.77 (95% CI: 0.83-0.97) for LOOCV testing. The same eleven-feature model was then used to predict recurrence, yielding a ROC-AUC of 0.92 (P = 0.0004) in the raw data set and 0.76 (95% CI: 0.64-0.87) for LOOCV testing. Conclusions: The RBM3 biomarker alone is a strong prognostic marker for the prediction of aggressive PCa and biochemical recurrence. Further, RBM3 appears to be down-regulated in aggressive and recurrent tumors.

High mobility group A1 (HMGA1) protein and gene expression correlate with ER-negativity and poor outcomes in breast cancer.

PURPOSE: The high mobility group A1 (HMGA1) chromatin remodeling protein is required for metastatic progression and cancer stem cell properties in preclinical breast cancer models, although its role in breast carcinogenesis has remained unclear. To investigate HMGA1 in primary breast cancer, we evaluated immunoreactivity score (IRS) in tumors from a large cohort of Asian women; HMGA1 gene expression was queried from two independent Western cohorts. METHODS: HMGA1 IRS was generated from breast tumors in Korean women as the product of staining intensity (weak = 1, moderate = 2, strong = 3) and percent positive cells (< 5% = 0, 5-30% = 1, 30-60% = 2, > 60% = 3), and stratified into three groups: low (< 3), intermediate (3-6), high (> 6). We assessed HMGA1 and estrogen receptor (ESR1) gene expression from two large databases (TCGA, METABRIC). Overall survival was ascertained from the METABRIC cohort. RESULTS: Among 540 primary tumors from Korean women (181 ER-negative, 359 ER-positive), HMGA1 IRS was < 3 in 89 (16.5%), 3-6 in 215 (39.8%), and > 6 in 236 (43.7%). High HMGA1 IRS was associated with estrogen receptor (ER)-negativity (χ2 = 12.07; P = 0.002) and advanced nuclear grade (χ2 = 12.83; P = 0.012). In two large Western cohorts, the HMGA1 gene was overexpressed in breast cancers compared to non-malignant breast tissue (P < 0.0001), including Asian, African American, and Caucasian subgroups. HMGA1 was highest in ER-negative tumors and there was a strong inverse correlation between HMGA1 and ESR1 gene expression (Pearson r = - 0.60, P < 0.0001). Most importantly, high HMGA1 predicted decreased overall survival (P < 0.0001) for all women with breast cancer and further stratified ER-positive tumors into those with inferior outcomes. CONCLUSIONS: Together, our results suggest that HMGA1 contributes to estrogen-independence, tumor progression, and poor outcomes. Moreover, further studies are warranted to determine whether HMGA1 could serve as a prognostic marker and therapeutic target for women with breast cancer.

Publisher Correction: 3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification.

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.

Advances in the computational and molecular understanding of the prostate cancer cell nucleus.

Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features, such as nuclear size and shape, texture, and spatial architecture, reflect the complex molecular-level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy-induced nuclear changes, can enable a detailed and objective analysis of the PCa cell nucleus. Recent advances in machine learning-based approaches can now automatically mine data related to these changes to aid in the diagnosis, decision making, and prediction of PCa prognoses. In this review, we use PCa as a case study to connect the molecular-level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we will discuss recent developments to our understanding of the molecular events that drive nuclear alterations in the context of PCa: the role of the nuclear matrix and lamina in size and shape changes, the role of 3-dimensional chromatin organization and epigenetic modifications in textural changes, and the role of the tumor microenvironment in altering nuclear spatial topology. We will then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision making, and predict potential outcomes, such as biochemical recurrence and survival. Finally, we will discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.

PBOV1 as a potential biomarker for more advanced prostate cancer based on protein and digital histomorphometric analysis.

BACKGROUND: There are few tissue-based biomarkers that can accurately predict prostate cancer (PCa) progression and aggressiveness. We sought to evaluate the clinical utility of prostate and breast overexpressed 1 (PBOV1) as a potential PCa biomarker. METHODS: Patient tumor samples were designated by Grade Groups using the 2014 Gleason grading system. Primary radical prostatectomy tumors were obtained from 48 patients and evaluated for PBOV1 levels using Western blot analysis in matched cancer and benign cancer-adjacent regions. Immunohistochemical evaluation of PBOV1 was subsequently performed in 80 cancer and 80 benign cancer-adjacent patient samples across two tissue microarrays (TMAs) to verify protein levels in epithelial tissue and to assess correlation between PBOV1 proteins and nuclear architectural changes in PCa cells. Digital histomorphometric analysis was used to track 22 parameters that characterized nuclear changes in PBOV1-stained cells. Using a training and test set for validation, multivariate logistic regression (MLR) models were used to identify significant nuclear parameters that distinguish Grade Group 3 and above PCa from Grade Group 1 and 2 PCa regions. RESULTS: PBOV1 protein levels were increased in tumors from Grade Group 3 and above (GS 4 + 3 and ≥ 8) regions versus Grade Groups 1 and 2 (GS 3 + 3 and 3 + 4) regions (P = 0.005) as assessed by densitometry of immunoblots. Additionally, by immunoblotting, PBOV1 protein levels differed significantly between Grade Group 2 (GS 3 + 4) and Grade Group 3 (GS 4 + 3) PCa samples (P = 0.028). In the immunohistochemical analysis, measures of PBOV1 staining intensity strongly correlated with nuclear alterations in cancer cells. An MLR model retaining eight parameters describing PBOV1 staining intensity and nuclear architecture discriminated Grade Group 3 and above PCa from Grade Group 1 and 2 PCa and benign cancer-adjacent regions with a ROC-AUC of 0.90 and 0.80, respectively, in training and test sets. CONCLUSIONS: Our study demonstrates that the PBOV1 protein could be used to discriminate Grade Group 3 and above PCa. Additionally, the PBOV1 protein could be involved in modulating changes to the nuclear architecture of PCa cells. Confirmatory studies are warranted in an independent population for further validation.

Nuclear Shape and Architecture in Benign Fields Predict Biochemical Recurrence in Prostate Cancer Patients Following Radical Prostatectomy: Preliminary Findings.

BACKGROUND: Gleason scoring represents the standard for diagnosis of prostate cancer (PCa) and assessment of prognosis following radical prostatectomy (RP), but it does not account for patterns in neighboring normal-appearing benign fields that may be predictive of disease recurrence. OBJECTIVE: To investigate (1) whether computer-extracted image features within tumor-adjacent benign regions on digital pathology images could predict recurrence in PCa patients after surgery and (2) whether a tumor plus adjacent benign signature (TABS) could better predict recurrence compared with Gleason score or features from benign or cancerous regions alone. DESIGN, SETTING, AND PARTICIPANTS: We studied 140 tissue microarray cores (0.6mm each) from 70 PCa patients following surgery between 2000 and 2004 with up to 14 yr of follow-up. Overall, 22 patients experienced recurrence (biochemical [prostate-specific antigen], local, or distant recurrence and cancer death) and 48 did not. INTERVENTION: RP was performed in all patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The top 10 features identified as most predictive of recurrence within both the benign and cancerous regions were combined into a 10-feature signature (TABS). Computer-extracted nuclear shape and architectural features from cancerous regions, adjacent benign fields, and TABS were evaluated via random forest classification accuracy and Kaplan-Meier survival analysis. RESULTS AND LIMITATIONS: Tumor-adjacent benign field features were predictive of recurrence (area under the receiver operating characteristic curve [AUC]: 0.72). Tumor-field nuclear shape descriptors and benign-field local nuclear arrangement were the predominant features found for TABS (AUC: 0.77). Combining TABS with Gleason sum further improved identification of recurrence (AUC: 0.81). All experiments were performed using threefold cross-validation without independent test set validation. CONCLUSIONS: Computer-extracted nuclear features within cancerous and benign regions predict recurrence following RP. Furthermore, TABS was shown to provide added value to common predictors including Gleason sum and Kattan and Stephenson nomograms. PATIENT SUMMARY: Future studies may benefit from evaluation of benign regions proximal to the tumor on surgically excised prostate cancer tissue for assessing risk of disease recurrence.

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

A Novel Quantitative Multiplex Tissue Immunoblotting for Biomarkers Predicts a Prostate Cancer Aggressive Phenotype.

BACKGROUND: Early prediction of disease progression in men with very low-risk (VLR) prostate cancer who selected active surveillance (AS) rather than immediate treatment could reduce morbidity associated with overtreatment. METHODS: We evaluated the association of six biomarkers [Periostin, (-5, -7) proPSA, CACNA1D, HER2/neu, EZH2, and Ki-67] with different Gleason scores and biochemical recurrence (BCR) on prostate cancer TMAs of 80 radical prostatectomy (RP) cases. Multiplex tissue immunoblotting (MTI) was used to assess these biomarkers in cancer and adjacent benign areas of 5 µm sections. Multivariate logistic regression (MLR) was applied to model our results. RESULTS: In the RP cases, CACNA1D, HER2/neu, and Periostin expression were significantly correlated with aggressive phenotype in cancer areas. An MLR model in the cancer area yielded a ROC-AUC = 0.98, whereas in cancer-adjacent benign areas, yielded a ROC-AUC = 0.94. CACNA1D and HER2/neu expression combined with Gleason score in a MLR model yielded a ROC-AUC = 0.79 for BCR prediction. In the small biopsies from an AS cohort of 61 VLR cases, an MLR model for prediction of progressors at diagnosis retained (-5, -7) proPSA and CACNA1D, yielding a ROC-AUC of 0.78, which was improved to 0.82 after adding tPSA into the model. CONCLUSIONS: The molecular profile of biomarkers is capable of accurately predicting aggressive prostate cancer on retrospective RP cases and identifying potential aggressive prostate cancer requiring immediate treatment on the AS diagnostic biopsy but limited in BCR prediction. IMPACT: Comprehensive profiling of biomarkers using MTI predicts prostate cancer aggressive phenotype in RP and AS biopsies.

Evaluation of two mitochondrial DNA biomarkers for prostate cancer detection.

BACKGROUND: A 3.4kb deletion (3.4kbΔ ) in mitochondrial DNA (mtDNA) found in histologically normal prostate biopsy specimens has been reported to be a biomarker for the increased probability of prostate cancer. Increased mtDNA copy number is also reported as associated with cancer. OBJECTIVE: Independent evaluation of these two potential prostate cancer biomarkers using formalin-fixed paraffin-embedded (FFPE) prostate tissue and matched urine and serum from a high risk cohort of men with and without prostate cancer. METHODS: Biomarker levels were detected via qPCR. RESULTS: Both 3.4kbΔ and mtDNA levels were significantly higher in cancer patient FFPE cores (p= 0.045 and p= 0.070 respectively at > 90% confidence). Urine from cancer patients contained significantly higher levels of mtDNA (p= 0.006, 64.3% sensitivity, 86.7% specificity). Combining the 3.4kbΔ and mtDNA gave better performance of detecting prostate cancer than either biomarker alone (FFPE 73.7% sensitivity, 65% specificity; urine 64.3% sensitivity, 100% specificity). In serum, there was no difference for any of the biomarkers. CONCLUSIONS: This is the first report on detecting the 3.4kbΔ in urine and evaluating mtDNA levels as a prostate cancer biomarker. A confirmation study with increased sample size and possibly with additional biomarkers would need to be conducted to corroborate and extend these observations.

Macrophage inhibitory cytokine 1 biomarker serum immunoassay in combination with PSA is a more specific diagnostic tool for detection of prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most common malignancy among men in the United States. Though highly sensitive, the often-used prostate-specific antigen (PSA) test has low specificity which leads to overdiagnosis and overtreatment of PCa. This paper presents results of a retrospective study that indicates that testing for macrophage inhibitory cytokine 1 (MIC-1) concentration along with the PSA assay could provide much improved specificity to the assay. METHODS: The MIC-1 serum level was determined by a novel p-Chip-based immunoassay run on 70 retrospective samples. The assay was configured on p-Chips, small integrated circuits (IC) capable of storing in their electronic memories a serial number to identify the molecular probe immobilized on its surface. The distribution of MIC-1 and pre-determined PSA concentrations were displayed in a 2D plot and the predictive power of the dual MIC-1/PSA assay was analyzed. RESULTS: MIC-1 concentration in serum was elevated in PCa patients (1.44 ng/ml) compared to normal and biopsy-negative individuals (0.93 ng/ml and 0.88 ng/ml, respectively). In addition, the MIC-1 level was correlated with the progression of PCa. The area under the receiver operator curve (AUC-ROC) was 0.81 providing an assay sensitivity of 83.3% and specificity of 60.7% by using a cutoff of 0.494 for the logistic regression value of MIC-1 and PSA. Another approach, by defining high-frequency PCa zones in a two-dimensional plot, resulted in assay sensitivity of 78.6% and specificity of 89.3%. CONCLUSIONS: The analysis based on correlation of MIC-1 and PSA concentrations in serum with the patient PCa status improved the specificity of PCa diagnosis without compromising the high sensitivity of the PSA test alone and has potential for PCa prognosis for patient therapy strategies.

The Upregulation of PI3K/Akt and MAP Kinase Pathways is Associated with Resistance of Microtubule-Targeting Drugs in Prostate Cancer.

Resistance is a significant limitation to the effectiveness of cancer therapies. The PI3K/Akt and MAP kinase pathways play important roles in a variety of normal cellular processes and tumorigenesis. This study is designed to explore the relationship of these signaling pathways with multidrug resistance in prostate cancer (PCa). The PI3K/Akt and MAP kinase pathways were investigated utilizing paclitaxel resistant DU145-TxR PCa cells and their parental non-resistant DU145 cells to determine their relationship with resistance to paclitaxel and other anticancer drugs. Our results demonstrate that the PI3K/Akt and MAP kinase pathways are upregulated in DU145-TxR cells compared to the DU145 cells. Inactivating these pathways using the PI3K/Akt pathway inhibitor LY294002 or the MAP kinase pathway inhibitor PD98059 renders the DU145-TxR cells more sensitive to paclitaxel. We investigated the effects of these inhibitors on other anticancer drugs including docetaxel, vinblastine, doxorubicin, 10-Hydroxycamptothecin (10-HCPT) and cisplatin and find that both inhibitors induces DU145-TxR cells to be more sensitive only to the microtubule-targeting drugs (paclitaxel, docetaxel and vinblastine). Furthermore, the treatment with these inhibitors induces cleaved-PARP production in DU145-TxR cells, suggesting that apoptosis induction might be one of the mechanisms for the reversal of drug resistance. In conclusion, the PI3K/Akt and MAP kinase pathways are associated with resistance to multiple chemotherapeutic drugs. Inactivating these pathways renders these PCa cells more sensitive to microtubule-targeting drugs such as paclitaxel, docetaxel and vinblastine. Combination therapies with novel inhibitors of these two signaling pathways potentially represents a more effective treatment for drug resistant PCa.

Epithelial-mesenchymal transition in prostate cancer is associated with quantifiable changes in nuclear structure.

BACKGROUND: Prostate cancer progression is concomitant with quantifiable nuclear structure and texture changes as compared to non-cancer tissue. Malignant progression is associated with an epithelial-mesenchymal transition (EMT) program whereby epithelial cancer cells take on a mesenchymal phenotype and dissociate from a tumor mass, invade, and disseminate to distant metastatic sites. The objective of this study was to determine if epithelial and mesenchymal prostate cancer cells have different nuclear morphology. METHODS: Murine tibia injections of epithelial PC3 (PC3-Epi) and mesenchymal PC3 (PC3-EMT) prostate cancer cells were processed and stained with H&E. Cancer cell nuclear image data was obtained using commercially available image-processing software. Univariate and multivariate statistical analysis were used to compare the two phenotypes. Several non-parametric classifiers were constructed and permutation-tested at various training set fractions to ensure robustness of classification between PC3-Epi and PC3-EMT cells in vivo. RESULTS: PC3-Epi and PC3-EMT prostate cancer cells were separable at the single cell level in murine tibia injections on the basis of nuclear structure and texture remodeling associated with an EMT. Support vector machine and multinomial logistic regression models based on nuclear architecture features yielded AUC-ROC curves of 0.95 and 0.96, respectively, in separating PC3-Epi and PC3-EMT prostate cancer cells in vivo. CONCLUSIONS: Prostate cancer cells that have undergone an EMT demonstrated an altered nuclear structure. The association of nuclear changes and a mesenchymal phenotype demonstrates quantitative morphometric image analysis may be used to detect cancer cells that have undergone EMT. This morphometric measurement could provide valuable prognostic information in patients regarding the likelihood of [future] metastatic disease.

Nuclear morphometry, epigenetic changes, and clinical relevance in prostate cancer.

Nuclear structure alterations in cancer involve global genetic (mutations, amplifications, copy number variations, translocations, etc.) and epigenetic (DNA methylation and histone modifications) events that dramatically and dynamically spatially change chromatin, nuclear body, and chromosome organization. In prostate cancer (CaP) there appears to be early (<50 years) versus late (>60 years) onset clinically significant cancers, and we have yet to clearly understand the hereditary and somatic-based molecular pathways involved. We do know that once cancer is initiated, dedifferentiation of the prostate gland occurs with significant changes in nuclear structure driven by numerous genetic and epigenetic processes. This review focuses upon the nuclear architecture and epigenetic dynamics with potential translational clinically relevant applications to CaP. Further, the review correlates changes in the cancer-driven epigenetic process at the molecular level and correlates these alterations to nuclear morphological quantitative measurements. Finally, we address how we can best utilize this knowledge to improve the efficacy of personalized treatment of cancer.

EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance.

Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

Hypermethylation of genes detected in urine from Ghanaian adults with bladder pathology associated with Schistosoma haematobium infection.

PURPOSE: Schistosoma haematobium is associated with chronic bladder damage and may subsequently induce bladder cancer in humans, thus posing a serious threat where the parasite is endemic. Here we evaluated aberrant promoter DNA methylation as a potential biomarker to detect severe bladder damage that is associated with schistosomiasis by analyzing urine specimens. MATERIALS AND METHODS: A quantitative methylation-specific PCR (QMSP) assay was used to examine the methylation status of seven genes (RASSF1A, RARß2, RUNX3, TIMP3, MGMT, P16, ARF) in 57 urine samples obtained from volunteers that include infected and uninfected by S. haematobium from an endemic region. The Fishers Exact Test and Logistic Regression analysis were used to evaluate the methylation status with bladder damage (as assessed by ultrasound examination) in subjects with S. haematobium infection. RESULTS: RASSF1A and TIMP3 were significant to predict severe bladder damage both in univariate (p = 0.015 and 0.023 respectively) and in multivariate (p = 0.022 and 0.032 respectively) logistic regression analysis. Area under the receiver operator characteristic curves (AUC-ROC) for RASSF1A and TIMP3 to predict severe bladder damage were 67.84% and 63.73% respectively. The combined model, which used both RASSF1A and TIMP3 promoter methylation, resulted in significant increase in AUC-ROC compared to that of TIMP3 (77.55% vs. 63.73%.29; p = 0.023). CONCLUSIONS: In this pilot study, we showed that aberrant promoter methylation of RASSF1A and TIMP3 are present in urine sediments of patients with severe bladder damage associated with S. haematobium infection and that may be used to develop non-invasive biomarker of S. haematobium exposure and early molecular risk assessmentof neoplastic transformation.

Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer.

Drug resistance is a major limitation to the successful treatment of advanced prostate cancer (PCa). Patients who have metastatic, castration-resistant PCa (mCRPC) are treated with chemotherapeutics. However, these standard therapy modalities culminate in the development of resistance. We established paclitaxel resistance in a classic, androgen-insensitive mCRPC cell line (DU145) and, using a suite of molecular and biophysical methods, characterized the structural and functional changes in vitro and in vivo that are associated with the development of drug resistance. After acquiring paclitaxel-resistance, cells exhibited an abnormal nuclear morphology with extensive chromosomal content, an increase in stiffness, and faster cytoskeletal remodeling dynamics. Compared with the parental DU145, paclitaxel-resistant (DU145-TxR) cells became highly invasive and motile in vitro, exercised greater cell traction forces, and formed larger and rapidly growing tumors in mouse xenografts. Furthermore, DU145-TxR cells showed a discrete loss of keratins but a distinct gain of ZEB1, Vimentin and Snail, suggesting an epithelial-to-mesenchymal transition. These findings demonstrate, for the first time, that paclitaxel resistance in PCa is associated with a trans-differentiation of epithelial cell machinery that enables more aggressive and invasive phenotype and portend new strategies for developing novel biomarkers and effective treatment modalities for PCa patients.

Nuclear morphometry, nucleomics and prostate cancer progression.

Prostate cancer (PCa) results from a multistep process. This process includes initiation, which occurs through various aging events and multiple insults (such as chronic infection, inflammation and genetic instability through reactive oxygen species causing DNA double-strand breaks), followed by a multistep process of progression. These steps include several genetic and epigenetic alterations, as well as alterations to the chromatin structure, which occur in response to the carcinogenic stress-related events that sustain proliferative signaling. Events such as evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis are readily observed. In addition, in conjunction with these critical drivers of carcinogenesis, other factors related to the etiopathogenesis of PCa, involving energy metabolism and evasion of the immune surveillance system, appear to be involved. In addition, when cancer spread and metastasis occur, the 'tumor microenvironment' in the bone of PCa patients may provide a way to sustain dormancy or senescence and eventually establish a 'seed and soil' site where PCa proliferation and growth may occur over time. When PCa is initiated and progression ensues, significant alterations in nuclear size, shape and heterochromatin (DNA transcription) organization are found, and key nuclear transcriptional and structural proteins, as well as multiple nuclear bodies can lead to precancerous and malignant changes. These series of cellular and tissue-related malignancy-associated events can be quantified to assess disease progression and management.