Cisplatin-induced increase in heregulin 1 and its attenuation by the monoclonal ErbB3 antibody seribantumab in bladder cancer.

Cisplatin-based combination chemotherapy is the foundation for treatment of advanced bladder cancer (BlCa), but many patients develop chemoresistance mediated by increased Akt and ERK phosphorylation. However, the mechanism by which cisplatin induces this increase has not been elucidated. Among six patient-derived xenograft (PDX) models of BlCa, we observed that the cisplatin-resistant BL0269 express high epidermal growth factor receptor, ErbB2/HER2 and ErbB3/HER3. Cisplatin treatment transiently increased phospho-ErbB3 (Y1328), phospho-ERK (T202/Y204) and phospho-Akt (S473), and analysis of radical cystectomy tissues from patients with BlCa showed correlation between ErbB3 and ERK phosphorylation, likely due to the activation of ERK via the ErbB3 pathway. In vitro analysis revealed a role for the ErbB3 ligand heregulin1-ß1 (HRG1/NRG1), which is higher in chemoresistant lines compared to cisplatin-sensitive cells. Additionally, cisplatin treatment, both in PDX and cell models, increased HRG1 levels. The monoclonal antibody seribantumab, that obstructs ErbB3 ligand-binding, suppressed HRG1-induced ErbB3, Akt and ERK phosphorylation. Seribantumab also prevented tumor growth in both the chemosensitive BL0440 and chemoresistant BL0269 models. Our data demonstrate that cisplatin-associated increases in Akt and ERK phosphorylation is mediated by an elevation in HRG1, suggesting that inhibition of ErbB3 phosphorylation may be a useful therapeutic strategy in BlCa with high phospho-ErbB3 and HRG1 levels.

Androgen receptor transcriptional activity is required for heregulin-1ß-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while in vivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. In vitro treatment with the ErbB3 ligand heregulin-1ß (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3 cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Androgen Receptor-Mediated Nuclear Transport of NRDP1 in Prostate Cancer Cells Is Associated with Worse Patient Outcomes.

To our knowledge, our group is the first to demonstrate that NRDP1 is located in the nucleus as well as the cytoplasm of CaP cells. Subcellular fractionation, immunohistochemistry, and immunofluorescence analysis combined with confocal microscopy were used to validate this finding. Subcellular fractionation followed by western blot analysis revealed a strong association between AR and NRDP1 localization when AR expression and/or cellular localization was manipulated via treatment with R1881, AR-specific siRNA, or enzalutamide. Transfection of LNCaP with various NRDP1 and AR constructs followed by immunoprecipitation confirmed binding of NRDP1 to AR is possible and determined that binding requires the hinge region of AR. Co-transfection with NRDP1 constructs and HA-ubiquitin followed by subcellular fractionation confirmed that nuclear NRDP1 retains its ubiquitin ligase activity. We also show that increased nuclear NRDP1 is associated with PSA recurrence in CaP patients (n = 162, odds ratio; 1.238, p = 0.007) and that higher levels of nuclear NRDP1 are found in castration resistant cell lines (CWR22Rv1 and PC3) compared to androgen sensitive cell lines (LNCaP and MDA-PCa-3B). The combined data indicate that NRDP1 plays a role in mediating CaP progression and supports further investigation of both the mechanism by which nuclear transport occurs and the identification of specific nuclear targets.

CCN3 Proteins as a diagnostic marker in osteosarcoma patients: A case control study.

BACKGROUND: Osteosarcoma is the most prevalent type of primary bone sarcoma and is the major cause of deaths associated with cancer in children and adolescents. Despite novel and innovative therapies, early diagnosis of the osteosarcoma is still critically needed. Our study aimed to analyse the CCN3 proteins as a diagnostic marker and correlate their expression level with the severity of primary osteosarcoma patients. METHODS: In this prospective case-control study, after ethical clearance and informed consent, a total of 35 cases with primary osteosarcoma and ten otherwise healthy controls were enroled according to our strict inclusion-exclusion criteria. Tissue samples were collected during biopsy procedures in suspected cases and in controls during bone grafting procedures. The CCN3 expression level was measured by the western blotting assay. The clinic-radiological examinations were done in cases and graded according to the AJCC classification. Comparisons of CCN3 expression were measured between cases and controls, followed by correlation of their expression level with severity/grade of osteosarcoma in cases. RESULTS: All the demographic parameters showed insignificant differences. The CCN3 protein expressions were significantly upregulated in tissue samples of osteosarcoma patients (cases) compared to controls. The mean difference (p<0.0001) in CCN3 protein expression between cases' and controls' bony tissues was significant but showed insignificant correlation with the different grades of osteosarcoma. CONCLUSIONS: The upregulated CCN3 protein expression in osteosarcoma tissue along with significant differential manifestation in accordance with different grades of osteosarcoma make CCN3 suitable for a potential diagnostic biomarker. However, the author recommends further extensive multi-centric collaborative studies to increase our study reliability and generalizability.

The p14ARF tumor suppressor restrains androgen receptor activity and prevents apoptosis in prostate cancer cells.

Prostate cancer (PCa) is characterized by a unique dependence on optimal androgen receptor (AR) activity where physiological androgen concentrations induce proliferation but castrate and supraphysiological levels suppress growth. This feature has been exploited in bipolar androgen therapy (BAT) for castrate resistant malignancies. Here, we investigated the role of the tumor suppressor protein p14ARF in maintaining optimal AR activity and the function of the AR itself in regulating p14ARF levels. We used a tumor tissue array of differing stages and grades to define the relationships between these components and identified a strong positive correlation between p14ARF and AR expression. Mechanistic studies utilizing CWR22 xenograft and cell culture models revealed that a decrease in AR reduced p14ARF expression and deregulated E2F factors, which are linked to p14ARF and AR regulation. Chromatin immunoprecipitation studies identified AR binding sites upstream of p14ARF. p14ARF depletion enhanced AR-dependent PSA and TMPRSS2 transcription, hence p14ARF constrains AR activity. However, p14ARF depletion ultimately results in apoptosis. In PCa cells, AR co-ops p14ARF as part of a feedback mechanism to ensure optimal AR activity for maximal prostate cancer cell survival and proliferation.

Methionine Adenosyltransferase 1a (MAT1A) Enhances Cell Survival During Chemotherapy Treatment and is Associated with Drug Resistance in Bladder Cancer PDX Mice.

Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Tumor relapse following chemotherapy treatment has long been a significant challenge towards completely curing cancer. We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize molecular mechanisms that contribute to relapse following drug treatment in advanced bladder cancer. Transcriptomic profiling of bladder cancer xenograft tumors by RNA-sequencing analysis, before and after relapse, following a 21-day cisplatin/gemcitabine drug treatment regimen identified methionine adenosyltransferase 1a (MAT1A) as one of the significantly upregulated genes following drug treatment. Survey of patient tumor sections confirmed elevated levels of MAT1A in individuals who received chemotherapy. Overexpression of MAT1A in 5637 bladder cancer cells increased tolerance to gemcitabine and stalled cell proliferation rates, suggesting MAT1A upregulation as a potential mechanism by which bladder cancer cells persist in a quiescent state to evade chemotherapy.

Dacomitinib, but not lapatinib, suppressed progression in castration-resistant prostate cancer models by preventing HER2 increase.

BACKGROUND: Despite overexpression of the ErbB (EGFR/HER2/ErbB3/ErbB4) family in castration-resistant prostate cancer (CRPC), some inhibitors of this family, including the dual EGFR/HER2 inhibitor lapatinib, failed in Phase II clinical trials. Hence, we investigated mechanisms of lapatinib resistance to determine whether alternate ErbB inhibitors can succeed. METHODS: The CWR22 human tumour xenograft and its CRPC subline 22Rv1 and sera from lapatinib-treated CRPC patients from a previously reported Phase II trial were used to study lapatinib resistance. Mechanistic studies were conducted in LNCaP, C4-2 and 22Rv1 cell lines. RESULTS: Lapatinib increased intratumoral HER2 protein, which encouraged resistance to this treatment in mouse models. Sera from CRPC patients following lapatinib treatment demonstrated increased HER2 levels. Investigation of the mechanism of lapatinib-induced HER2 increase revealed that lapatinib promotes HER2 protein stability, leading to membrane localisation, EGFR/HER2 heterodimerisation and signalling, elevating cell viability. Knockdown of HER2 and ErbB3, but not EGFR, sensitised CRPC cells to lapatinib. At equimolar concentrations, the recently FDA-approved pan-ErbB inhibitor dacomitinib decreased HER2 protein stability, prevented ErbB membrane localisation (despite continued membrane integrity) and EGFR/HER2 heterodimerisation, thereby decreasing downstream signalling and increasing apoptosis. CONCLUSIONS: Targeting the EGFR axis using the irreversible pan-ErbB inhibitor dacomitinib is a viable therapeutic option for CRPC.

XPO1 inhibition by selinexor induces potent cytotoxicity against high grade bladder malignancies.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.

Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer.

Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

Enhancing the effectiveness of androgen deprivation in prostate cancer by inducing Filamin A nuclear localization.

As prostate cancer (CaP) is regulated by androgen receptor (AR) activity, metastatic CaP is treated with androgen deprivation therapy (ADT). Despite initial response, patients on ADT eventually progress to castration-resistant CaP (CRPC), which is currently incurable. We previously showed that cleavage of the 280 kDa structural protein Filamin A (FlnA) to a 90 kDa fragment, and nuclear localization of the cleaved product, sensitized CRPC cells to ADT. Hence, treatment promoting FlnA nuclear localization would enhance androgen responsiveness. Here, we show that FlnA nuclear localization induced apoptosis in CRPC cells during ADT, identifying it as a treatment tool in advanced CaP. Significantly, the natural product genistein combined polysaccharide (GCP) had a similar effect. Investigation of the mechanism of GCP-induced apoptosis showed that GCP induced FlnA cleavage and nuclear localization and that apoptosis resulting from GCP treatment was mediated by FlnA nuclear localization. Two main components of GCP are genistein and daidzein: the ability of GCP to induce G2 arrest was due to genistein whereas sensitivity to ADT stemmed from daidzein; hence, both were needed to mediate GCP's effects. FlnA cleavage is regulated by its phosphorylation; we show that ADT enhanced FlnA phosphorylation, which prevented its cleavage, whereas GCP inhibited FlnA phosphorylation, thereby sensitizing CaP cells to ADT. In a mouse model of CaP recurrence, GCP, but not vehicle, impeded relapse following castration, indicating that GCP, when administered with ADT, interrupted the development of CRPC. These results demonstrate the efficacy of GCP in promoting FlnA nuclear localization and enhancing androgen responsiveness in CaP.

Health related quality of life assessment in Pakistani paediatric cancer patients using PedsQL™ 4.0 generic core scale and PedsQL™ cancer module.

BACKGROUND: The purpose of the study was to evaluate and compare the HRQOL of paediatric cancer in comparison to the healthy children across age groups, using PedsQLTM 4.0 Generic Core Scales and the PedsQL™ Cancer Module. METHOD: The PedsQLTM 4.0 Generic Core Scales and PedsQL Cancer Module 3.0 were administered on 56 children including 26 cancer patients and 30 healthy children while employing self and proxy report forms. Furthermore, the results were compared with their healthy comparison group. RESULTS: The results indicated a significant relationship between HRQOL reports of cancer patients and their parents. However, the mean of paediatric cancer patients is significantly lower as compare to their healthy comparison group. The mean of proxy report is lower overall on both PedsQL and PedsQL cancer module reports. CONCLUSION: Conclusively, overall HRQOL of cancer patients was lower than healthy children but it is quite similar to their parents' perception. Whereas, the parental mean on PedsQL and PedsQL 3.0 Cancer Module are significantly low. The study indicated a marked difference between cancer patients and healthy children's HRQOL perception and unfortunately in country like Pakistan where cancer is on increase, no significant work has yet been done to explore this area of research. The present study highlighted the need to focus on the particular psychological health services required to serve the physically challenged population.

Nrdp1-mediated regulation of ErbB3 expression by the androgen receptor in androgen-dependent but not castrate-resistant prostate cancer cells.

Patients with advanced prostate cancer (PCa) are initially susceptible to androgen withdrawal (AW), but ultimately develop resistance to this therapy (castration-resistant PCa, CRPC). Here, we show that AW can promote CRPC development by increasing the levels of the receptor tyrosine kinase ErbB3 in androgen-dependent PCa, resulting in AW-resistant cell cycle progression and increased androgen receptor (AR) transcriptional activity. CRPC cell lines and human PCa tissue overexpressed ErbB3, whereas downregulation of ErbB3 prevented CRPC cell growth. Investigation of the mechanism by which AW augments ErbB3, using normal prostate-derived pRNS-1-1 cells, and androgen-dependent PCa lines LNCaP, PC346C, and CWR22 mouse xenografts, revealed that the AR suppresses ErbB3 protein levels, whereas AW relieves this suppression, showing for the first time the negative regulation of ErbB3 by AR. We show that AR activation promotes ErbB3 degradation in androgen-dependent cells, and that this effect is mediated by AR-dependent transcriptional upregulation of neuregulin receptor degradation protein-1 (Nrdp1), an E3 ubiquitin ligase that targets ErbB3 for degradation but whose role in PCa has not been previously examined. Therefore, AW decreases Nrdp1 expression, promoting ErbB3 protein accumulation, and leading to AR-independent proliferation. However, in CRPC sublines of LNCaP and CWR22, which strongly overexpress the AR, ErbB3 levels remain elevated due to constitutive suppression of Nrdp1, which prevents AR regulation of Nrdp1. Our observations point to a model of CRPC development in which progression of PCa to castration resistance is associated with the inability of AR to transcriptionally regulate Nrdp1, and predict that inhibition of ErbB3 during AW may impair CRPC development.

Nuclear versus cytoplasmic localization of filamin A in prostate cancer: immunohistochemical correlation with metastases.

PURPOSE: We previously showed that nuclear localization of the actin-binding protein, filamin A (FlnA), corresponded to hormone-dependence in prostate cancer. Intact FlnA (280 kDa, cytoplasmic) cleaved to a 90 kDa fragment which translocated to the nucleus in hormone-naïve cells, whereas in hormone-refractory cells, FlnA was phosphorylated, preventing its cleavage and nuclear translocation. We have examined whether FlnA localization determines a propensity to metastasis in advanced androgen-independent prostate cancer. EXPERIMENTAL DESIGN: We examined, by immunohistochemistry, FlnA localization in paraffin-embedded human prostate tissue representing different stages of progression. Results were correlated with in vitro studies in a cell model of prostate cancer. RESULTS: Nuclear FlnA was significantly higher in benign prostate (0.6612 +/- 0.5888), prostatic intraepithelial neoplasia (PIN; 0.6024 +/- 0.4620), and clinically localized cancers (0.69134 +/- 0.5686) compared with metastatic prostate cancers (0.3719 +/- 0.4992, P = 0.0007). Cytoplasmic FlnA increased from benign prostate (0.0833 +/- 0.2677), PIN (0.1409 +/- 0.2293), localized cancers (0.3008 +/- 0.3762, P = 0.0150), to metastases (0.7632 +/- 0.4414, P < 0.00001). Logistic regression of metastatic versus nonmetastatic tissue yielded the area under the receiver operating curve as 0.67 for nuclear-FlnA, 0.79 for cytoplasmic-FlnA, and 0.82 for both, indicating that metastasis correlates with cytoplasmic to nuclear translocation. In vitro studies showed that cytoplasmic localization of FlnA induced cell invasion whereas nuclear translocation of the protein inhibited it. FlnA dephosphorylation with the protein kinase A inhibitor H-89 facilitated FlnA nuclear translocation, resulting in decreased invasiveness and AR transcriptional activity, and induced sensitivity to androgen withdrawal in hormone-refractory cells. CONCLUSIONS: The data presented in this study indicate that in prostate cancer, metastasis correlates with cytoplasmic localization of FlnA and may be prevented by cleavage and subsequent nuclear translocation of this protein.