The androgen receptor (AR) plays a key role in progression to incurable androgen ablation-resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand-binding domain (designated as AR3, AR4, and AR5) in hormone-insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active, and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly up-regulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells, whereas specific knockdown of AR3 expression (without altering AR level) in hormone-resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct, yet essential, role in ablation-independent growth through the regulation of a unique set of genes, including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation independence during PCA progression, and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available antiandrogen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA. [Cancer Res 2009;69(6):2305-13]

Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue.

Summary The androgen receptor (AR) plays a critical role in prostate cancer. We have identified an ubiquitin E3 ligase RNF6 as one of AR-associated proteins in a proteomic screen. RNF6 induces AR ubiquitination and promotes AR transcriptional activity. Specific knockdown of RNF6 or mutation of RNF6-induced ubiquitination acceptor sites on AR selectively alters expression of a subset of AR target genes and diminishes recruitment of AR and its coactivators to androgen-responsive elements present in the regulatory region of these genes. Furthermore, RNF6 is overexpressed in human hormone-refractory prostate cancer tissues and required for prostate cancer cell growth under androgen-depleted conditions. Our data suggest that RNF6-induced ubiquitination may regulate AR transcriptional activity and specificity through modulating co-factor recruitment.

Drug resistance remains a clinical challenge in cancer treatment due to poor understanding of underlying mechanisms. We have established several drug-resistant prostate cancer cell lines by long-term culture in medium containing chemotherapeutic drugs. These resistant lines displayed a significant increase in side population cells due to overexpression of drug efflux pumps including ABCG2/BCRP and MDR1/Pgp. To uncover potential mechanisms underlying drug resistance, we performed microarray analysis to identify differentially expressed genes in 2 drug-resistant lines. We observed that POU5F1/OCT4, a transcription factor key to regulating pluripotency in embryonic stem cells, was upregulated in drug-resistant lines and accompanied by transcriptional activation of a set of its known target genes. Upregulation of OCT4 in drug-resistant cells was validated by RT-PCR and sequencing of PCR products as well as confirmation by Western blot and specific shRNA knockdown. Analysis of the regulatory region of POU5F1/OCT4 revealed a reduction of methylation in drug-resistant cell lines. Furthermore, these drug-resistant cells exhibited a significant increase in tumorigenicity in vivo. Subcutaneous inoculation of as few as 10 drug-resistant cells could initiate tumor formation in SCID mice, whereas no detectable tumors were observed from the parental line under similar conditions, suggesting that these drug-resistant cells may be enriched for tumor-initiating cells. Knocking down OCT4 expression by specific shRNAs attenuated growth of drug-resistant cells. Our data suggest that OCT4 re-expression in cancer cells may play an important role in carcinogenesis and provide one possible mechanism by which cancer cells acquire/maintain a drug-resistant phenotype.

Background: AR splice variants may play a critical role in prostate cancer. Results: AR3 modulates expression of tumor-promoting growth factors and promotes epithelial-mesenchymal transition, leading to development of prostatic intraepithelial neoplasia. Conclusion: AR3 promotes prostate cancer by modulating multiple tumor-associated autocrine/paracrine factors. Significance: Our findings provide new insights into mechanisms by which AR3 contributes to prostate cancer despite its heterogeneous expression pattern.

Our results suggest that p-STAT3 is an important factor during carcinogenesis and metastasis of lung carcinoma, and its relationship to EGFR mutation status may provide potential targeting opportunities in future therapies.

Despite the increasing incidence of combined small-cell lung cancer (C-SCLC) in recent years, there have not been many data on clinical prognostic factors predicting prognosis of C-SCLC patients. In present study, we sought pretreatment features especially basic laboratory parameters predicting survival of C-SCLC. We analyzed 613 small-cell lung cancer (SCLC) patients at our institution between January 2005 and December 2010. We identified 114 patients with C-SCLC. The pathologic and clinical characteristics of these patients were reviewed. Data of laboratory parameters obtained during regular examinations at diagnosis of these patients were examined. The Kaplan-Meier method was used to calculate the survival rate and depict the survival curves. The Cox regression model was used to analyze the independent factors affecting the overall survival (OS). These data were compared with the results obtained from our 499 pure SCLC patients who presented during the same time period. Of the 613 SCLC patients analyzed, 18.6 % of the patients presented with C-SCLC. No difference in OS was observed in patients with C-SCLC and patients with pure SCLC (P = 0.995). The Kaplan-Meier survival curves revealed that poor ECOG-PS (P < 0.001), extensive disease (P < 0.001), pathologic subtype of SC/LC (P < 0.001), not receiving surgery (P = 0.001), elevated serum lactate dehydrogenase (LDH) (P = 0.005), elevated NSE (P = 0.043), and elevated neutrophile-lymphocyte ratio (NLR) (P = 0.018) were associated with adverse prognosis of patients with C-SCLC. By multivariate analysis, OS was affected by ECOG-PS (hazard ratio 2.001, P = 0.012), disease extent (hazard ratio 3.406, P < 0.001), and NLR (hazard ratio 1.704, P = 0.030) in C-SCLC patients, while the risk factors that influenced the prognosis of the patients with pure SCLC were ECOG-PS (hazard ratio 2.132, P < 0.001), disease extent (hazard ratio 1.482, P < 0.001), and LDH (hazard ratio 1.811, P < 0.001). Patients with C-SCLC carry a similar prognosis than those with pure small-cell variety. Easily accessible pretreatment parameters such as NLR should be considered in defining the prognosis of C-SCLC patients besides disease extent and performance status.

Abstract. The aim of the present study was to investigate the prognostic value of the combined platelet (PLT), fibrinogen (FBG), neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) (CO-NPF) for postoperative outcomes in patients with lung adenosquamous cancer (ASC). Test results from patients who presented at The Cancer Institute and Hospital of Tianjin Medical University between January 2005 and December 2013 were retrospectively reviewed. CO-NPF was scored between 0 and 4 according to increased PLT, FBG, NLR and PLR prior to being split into two groups based on the presence (≥2) or absence (<2) of the combination of increased inflammatory indexes. In total, data from 134 patients with ASC were reviewed for the present study. Multivariate analysis identified that increased CO-NPF (P= 0.001 and P<0.001, respectively), PLR (P=0.011 and P=0.001, respectively) and FBG (P=0.001 and P<0.001, respectively) were independently associated with shorter disease-free survival (DFS) and overall survival (OS). NLR (P=0.006) and PLT (P=0.001) were independent prognostic factors for OS. The area under the receiver operating characteristic curves of CO-NPF (area under the curve, 0.652, P= 0.008, 95% confidence interval, 0.551-0.752) was increased compared with NLR, PLR, PLT and FBG individually, suggesting that CO-NPF has greater predictive value. CO-NPF was significantly and independently associated with shorter DFS and OS, and had greater predictive value compared with NLR, PLR, PLT and FBG in patients with ASC who underwent surgery.