Immunohistochemical analysis of RANKL in C4-2 tumored tibiae of control, prevention, and treatment groups

<p><b>Copyright information:</b></p><p>Taken from "Host-derived RANKL is responsible for osteolysis in a C4-2 human prostate cancer xenograft model of experimental bone metastases"</p><p>http://www.biomedcentral.com/1471-2407/7/148</p><p>BMC Cancer 2007;7():148-148.</p><p>Published online 3 Aug 2007</p><p>PMCID:PMC2034387.</p><p></p> Paraffin-embedded C4-2 tumored tibiae of the SCID mice were sectioned and stained for RANKL (negative control staining is shown in inset box). The tumor cells stained positively for RANKL. : Blood was collected to determine serum PSA levels by IMx Total PSA assay. Serum PSA levels were not significantly different between the C4-2 control with PBS injections (control), C4-2 with a concomitant subcutaneous injection of huRANKL MAb (5 mg/kg once a week) at implantation (prevention group), or C4-2 with a subcutaneous injection of huRANKL MAb (5 mg/kg biweekly, starting 3 weeks after implantation of C4-2 cells) (treatment group). Results are presented as mean ± SE

Methylation profiling identified novel differentially methylated markers including <i>OPCML</i> and <i>FLRT2</i> in prostate cancer

<p>To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including <i>OPCML, ELAVL2, EXT1, IRX5</i>, and <i>FLRT2</i>, were validated by pyrosequencing using the discovery cohort. <i>OPCML</i> and <i>FLRT2</i> were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, <i>OPCML</i>-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.</p

AR staining profiles of normal prostate, primary PCa and CRPC.

<p>(<b>A</b>) IHC staining for N- and C-terminal AR in normal prostate (NP) (a and b), hyperplastic prostate (HP) (c and d) and primary PCa (e-h) (magnification x200). (<b>B</b>) Comparison of AR staining profiles among normal prostate, hyperplastic prostate and primary PCa. (<b>C</b>) Comparison of AR staining profiles between primary PCa and metastatic CRPC.</p

Expression of AR variants and AR regulated proteins in metastatic CRPC.

<p>(<b>A</b>) IHC staining for N-terminal AR (a), C-terminal AR (b), PSA (c), PSMA (d), TMPRSS2 (e), AKT-1 (f), Ki-67(g), Negative control (h) on a metastatic CRPC tissue (magnification x200, insert x400). (<b>B</b>) PSA, PSMA, TMPRSS2 and AKT-1 staining profiles of CRPC.</p

The heterogeneity of AR expression in individual patients.

<p>Multiple metastatic sites of 42 CRPC patients had been analyzed by IHC using 2 AR antibodies. The staining results were summarized as N+C+ (blue), N+C↓ (orange) and N-C- (red). LN = lymph node; L =  lumbar vertebra; R. =  right; L. =  left; T =  thoracic vertebra.</p

Clinical data of 42 CRPC patients^*.

<p>*All 42 patients had castrate resistant prostate cancer at the time of autopsy, defined by the presence of a rising serum PSA following medical or surgical castration. All patients' tissues were obtained at autopsy under University of Washington Medical Center Prostate Cancer Donor Rapid Autopsy Program.</p

Comparison of IHC with RT-PCR results in PCa Metastases.

<p>+Intense expression, ±Limited/weak expression, − No expression.</p

Antibodies used in this study.

<p>Antibodies used in this study.</p

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

<div><p>Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. <i>In vivo</i>, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease. </p> </div

Cabozantinib attenuates bone responses to tumor and increases normal bone volume in tumor unaffected areas.

<p>A. LuCaP 23.1 and C4-2B cell growth in tibiae causes large increases in trabecular bone volume. µCT images show that cabozantinib alleviates the bone response to both tumors. In LuCaP 23.1 tumored tibiae cabozantinib caused decreases in BV, while increases in BV were detected in C4-2B tumored tibiae of cabozantinib-treated animals vs control-tumored tibiae. The overall effects are combination of abolishment of tumor effects on the bone as well as cabozantinib effects on normal bone. Details of the effects are provided in Table 1. B. Analysis of non-tumored contralateral tibiae of the experimental animals shows that treatment with cabozantinib results in increased bone volume in both intact and castrated male mice. C. <i>In </i><i>vitro</i>, cabozantinib treatment inhibits proliferation of MC3T3 pre-osteoblast cells in a concentration-dependent manner, while promoting ALP activity and mineralization. Fold change in cells response measures was estimated from a single experiment that was repeated three times, and association with cabozantinib concentration was quantified and tested using linear regression models.</p