Xiangtian Kong

A Novel Androgen Receptor Splice Variant Is Up-regulated during Prostate Cancer Progression and Promotes Androgen Depletion–Resistant Growth

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.

Regulation of androgen receptor transcriptional activity and specificity by RNF6-induced ubiquitination.

The androgen receptor (AR) plays a critical role in prostate cancer. We have identified a ubiquitin E3 ligase, RNF6, as an AR-associated protein 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 hormone-refractory human 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 cofactor recruitment.

Prostate Cancer: Comparison of 3D T2-Weighted With Conventional 2D T2-Weighted Imaging for Image Quality and Tumor Detection

OBJECTIVE The purpose of this study was to compare a 3D T2-weighted imaging sequence with a conventional multiplanar 2D turbo spin-echo T2-weighted sequence in terms of tumor detection and staging of prostate cancer, as well as image quality. MATERIALS AND METHODS Before prostatectomy, 38 men (mean age, 60 years) with prostate cancer underwent MRI of the prostate with multiplanar 2D turbo spin-echo T2-weighted sequences (total acquisition time, approximately 11 minutes 4 seconds) and a 3D T2-weighted sampling perfection with application optimized contrasts sequence with different flip angle evolutions (SPACE) (acquisition time, approximately 3 minutes 52 seconds). Two blinded observers in consensus reviewed 2D turbo spin-echo T2-weighted images and SPACE images for detection of peripheral zone cancer, extracapsular extension, and seminal vesicle invasion. The observers also assessed subjective image quality and measured the signal-to-noise ratio (SNR) of normal peripheral zone and tumor-to-peripheral zone contrast. Prostatectomy was used as the reference standard. The diagnostic accuracy of the two sequences was assessed with generalized estimating equations and McNemar tests. The agreement between sequences was assessed with kappa coefficients. A paired Wilcoxon signed rank test was used to compare the subjective image quality, SNR, and tumor-to-peripheral zone contrast of the two sequences. RESULTS For tumor detection and diagnosis of extracapsular extension, there was substantial agreement between the two sequences (kappa = 0.79, kappa = 0.76) with no difference in sensitivity, specificity, positive predictive value, negative predictive value, accuracy (p = 0.25-1), or image quality (p = 0.937). Images obtained with the 2D turbo spin-echo sequence had a significantly higher SNR ratio for normal peripheral zone (p = 0.0010), but SPACE images had significantly greater tumor-to-peripheral zone contrast (p < 0.0001). CONCLUSION In comparison with conventional multiplanar 2D turbo spin-echo MRI of the prostate, 3D T2-weighted SPACE MRI was associated with substantial time saving (nearly 8 minutes), had similar image quality and accuracy in the diagnosis of tumor and extracapsular extension, and had better tumor conspicuity.

Prostate cancer vs. post-biopsy hemorrhage: diagnosis with T2- and diffusion-weighted imaging.

To assess the value of quantitative T2 signal intensity (SI) and apparent diffusion coefficient (ADC) to differentiate prostate cancer from post‐biopsy hemorrhage, using prostatectomy as the reference.

Distinct nuclear and cytoplasmic functions of androgen receptor cofactor p44 and association with androgen-independent prostate cancer

Androgen receptor (AR) mediates transcriptional activation of diverse target genes through interactions with various coactivators that may alter its function and help mediate the switch between prostate cell proliferation and differentiation. We recently identified p44/MEP50 as an AR coactivator and further showed that it is expressed primarily in the nucleus and cytoplasm of benign prostate epithelial and prostate cancer cells, respectively. We also showed that haploinsufficiency in p44+/− mice causes prostate epithelial cell proliferation. To establish direct cause-and-effect relationships, we have used p44 fusion proteins that are selectively expressed in the nucleus or cytoplasm of prostate cancer cells (LNCaP), along with RNAi analyses, to examine effects of p44 both in vitro and in vivo (in tumor xenografts). We show that preferential expression of p44 in the nucleus inhibits proliferation of LNCaP cells in an AR-dependent manner, whereas preferential expression of p44 in the cytoplasm enhances cell proliferation. These effects appear to be mediated, at least in part, through the regulation of distinct cell-cycle regulatory genes that include p21 (up-regulated by nuclear p44) and cyclin D2 and CDK6 (up-regulated by cytoplasmic p44). Importantly, we also demonstrate that altered p44 expression is associated with androgen-independent prostate cancer. Our results indicate that nuclear p44 and cytoplasmic p44 have distinct and opposing functions in the regulation of prostate cancer cell proliferation.

Compensatory Upregulation of Tyrosine Kinase Etk/BMX in Response to Androgen Deprivation Promotes Castration-Resistant Growth of Prostate Cancer Cells

We previously showed that targeted expression of non-receptor tyrosine kinase Etk/BMX in mouse prostate induces prostate intraepithelial neoplasia, implying a possible causal role of Etk in prostate cancer development and progression. Here, we report that Etk is upregulated in both human and mouse prostates in response to androgen ablation. Etk expression seems to be differentially regulated by androgen and interleukin 6 (IL-6), which is possibly mediated by the androgen receptor (AR) in prostate cancer cells. Our immunohistochemical analysis of tissue microarrays containing 112 human prostate tumor samples revealed that Etk expression is elevated in hormone-resistant prostate cancer and positively correlated with tyrosine phosphorylation of AR (Pearson correlation coefficient rho = 0.71, P < 0.0001). AR tyrosine phosphorylation is increased in Etk-overexpressing cells, suggesting that Etk may be another tyrosine kinase, in addition to Src and Ack-1, which can phosphorylate AR. We also showed that Etk can directly interact with AR through its Src homology 2 domain, and such interaction may prevent the association of AR with Mdm2, leading to stabilization of AR under androgen-depleted conditions. Overexpression of Etk in androgen-sensitive LNCaP cells promotes tumor growth while knocking down Etk expression in hormone-insensitive prostate cancer cells by a specific shRNA that inhibits tumor growth under androgen-depleted conditions. Taken together, our data suggest that Etk may be a component of the adaptive compensatory mechanism activated by androgen ablation in prostate and may play a role in hormone resistance, at least in part, through direct modulation of the AR signaling pathway.

Tyrosine kinase Etk/BMX is up-regulated in human prostate cancer and its overexpression induces prostate intraepithelial neoplasia in mouse

The nonreceptor tyrosine kinase Etk/BMX was originally identified from the human prostate xenograft CWR22. Here, we report that Etk is up-regulated in human prostate tumor specimens surveyed. Knocking down Etk expression by a specific small interfering RNA (siRNA) in prostate cancer cells attenuates cell proliferation, suggesting an essential role of Etk for prostate cancer cell survival and growth. Targeted expression of Etk in mouse prostate epithelium results in pathologic changes resembling human prostatic intraepithelial neoplasia, indicating that up-regulation of Etk may contribute to prostate cancer development. A marked increase of luminal epithelial cell proliferation was observed in the Etk transgenic prostate, which may be attributed in part to the elevated activity of Akt and signal transducers and activators of transcription 3 (STAT3). More interestingly, the expression level of acetyltransferase cyclic AMP-responsive element binding protein-binding protein (CBP) is also increased in the Etk transgenic prostate as well as in a prostate cancer cell line overexpressing Etk, concomitant with elevated histone 3 acetylation at lysine 18 (H3K18Ac). Down-modulation of Etk expression by a specific siRNA leads to a decrease of H3 acetylation in prostate cancer cell lines. Our data suggest that Etk may also modulate chromatin remodeling by regulating the activity of acetyltransferases, such as CBP. Given that Etk may exert its effects in prostate through modulation of multiple signaling pathways altered in human prostate cancer, the Etk transgenic mouse model may be a useful tool for studying the functions of Etk and identification of new molecular markers and drug targets relevant to human diseases.

Prostate cancer: utility of fusion of T2-weighted and high b-value diffusion-weighted images for peripheral zone tumor detection and localization.

To retrospectively assess the utility of fusion of T2‐weighted images (T2WI) and high b‐value diffusion‐weighted images (DWI) for prostate cancer detection and localization.

Midkine is a NF-κB-inducible gene that supports prostate cancer cell survival

BackgroundMidkine is a heparin-binding growth factor that is over-expressed in various human cancers and plays important roles in cell transformation, growth, survival, migration, and angiogenesis. However, little is known about the upstream factors and signaling mechanisms that regulate midkine gene expression.MethodsTwo prostate cancer cell lines LNCaP and PC3 were studied for their expression of midkine. Induction of midkine expression in LNCaP cells by serum, growth factors and cytokines was determined by Western blot analysis and/or real-time quantitative reverse-transcription – polymerase chain reaction (RT-PCR). The cell viability was determined by the trypan blue exclusion assay when the LNCaP cells were treated with tumor necrosis factor alpha (TNFα) and/or recombinant midkine. When the LNCaP cells were treated with recombinant midkine, activation of intracellular signalling pathways was determined by Western blot analysis. Prostate tissue microarray slides containing 129 cases (18 normal prostate tissues, 40 early stage cancers, and 71 late stage cancers) were assessed for midkine expression by immunohistochemical staining.ResultsWe identified that fetal bovine serum, some growth factors (epidermal growth factor, androgen, insulin-like growth factor-I, and hepatocyte growth factor) and cytokines (TNFα and interleukin-1beta) induced midkine expression in a human prostate cancer cell line LNCaP cells. TNFα also induced midkine expression in PC3 cells. TNFα was the strongest inducer of midkine expression via nuclear factor-kappa B pathway. Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. Knockdown of endogenous midkine expression by small interfering RNA enhanced TNFα-induced apoptosis in LNCaP cells. Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells. Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFα in advanced prostate cancer.ConclusionThese findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFα, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer.

Prostate Cancer: Comparison of Tumor Visibility on Trace Diffusion- Weighted Images and the Apparent Diffusion Coefficient Map

OBJECTIVE The purpose of our study was to compare the visibility of prostate cancer on trace diffusion-weighted (DW) images and the apparent diffusion coefficient (ADC) map. MATERIALS AND METHODS In this retrospective study, 45 patients with prostate cancer underwent preoperative MRI, including DW imaging (DWI) (b values 0, 500, and 1,000 s/mm(2)). A single observer reviewed the images in conjunction with tumor maps constructed from prostatectomy. For 132 peripheral zone (PZ) tumor foci, the visibility and contrast relative to benign PZ were recorded for T2-weighted imaging, trace DWI b500 images, trace DWI b1,000 images, and ADC maps. Trace DWI b1,000 images and ADC maps were compared in terms of Gleason score, size, normalized T2 signal intensity, ADC, and normalized ADC of visible tumors. RESULTS For each image set, the percentage of visible tumor foci and contrast relative to benign PZ were as follows: T2-weighted imaging, 80.3% and 0.411; trace DWI b500, 26.5% and 0.131; trace DWI b1,000, 46.2% and 0.119; and ADC maps, 62.1% and 0.309. Forty-seven tumor foci were visible on both trace DWI b1,000 images and ADC maps, 14 only on trace DWI b1,000 images, 35 only on ADC maps, and 36 on neither image set. There was no significant difference in Gleason score, size, normalized T2 signal intensity, ADC, or normalized ADC between tumors visible only on trace DWI b1,000 images and those visible only on ADC maps. CONCLUSION Given a greater proportion of tumors visible on the ADC map than trace DWI and greater contrast relative to benign PZ on the ADC map, we suggest that, when performing DWI of the prostate, careful attention be given to the ADC map for tumor identification.