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

Expression of MET, P-MET, and VEGFR2 in primary and metastatic patient samples.

<p>IHC and analyses were performed as described in the Methods section. Graphical profiles illustrating distributions of staining intensity were constructed by calculating simple averages across all non-missing sections in each staining category. In each site, the mean staining index is marked by a filled orange circle and orange bars represent 95% CIs. Representative examples of staining are shown for each protein. A. MET is strongly expressed in both primary and metastatic PCa, though it is significantly increased in BM and decreased in soft tissue metastases vs. primary PCa. B. P-MET levels are higher in BM, LN and other soft tissue metastases, while no alteration was detected in liver metastases when compared to primary PCa. C. VEGFR2 expression is significantly increased across PCa metastatic lesions as compared to primary PCa. Images were taken at 400 x magnification.</p

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

Cabozantinib (60 mg/kg) inhibits tumor growth in androgen-sensitive and castration-resistant PCa in bone.

<div><p>A. Levels of cabozantinib receptors in LuCaP 23.1 and C4-2B subcutaneous tumors. qPCR was used on RNA isolated from subcutaneous tumors to determine expression levels of MET, VEGFR2m, AXL, RET and KIT. To calibrate the signal we used four-fold dilution of LNCaP cDNA (RET), PC-3 cDNA (MET, AXL, KIT) and LuCaP 23.1 (VEGFR2m). Selection of the calibrator cDNA was based on signal for each specific message. Signal was normalized to housekeeping gene RPL13a. Our results indicate that LuCaP 23.1 tumors express all of the cabozantinib targets and C4-2B tumors express VEGFR2m, AXL and RET, low levels of MET, and no KIT. In these qPCR experiments, we measured relative levels of the target transcripts and not their actual numbers, therefore we cannot compare expression levels of the different targets to each other, and comment whether RET, which gave the higher signal, might be expressed at higher copy number vs MET, and therefore is more important in these models. </p> <p>B. Linear models of PSA growth show that cabozantinib decreases PSA levels in both androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B models. C. BrdU staining of tibiae shows that cabozantinib decreases proliferation of androgen-sensitive and castration-resistant tumor cells in bone, 2-sided t-test was used to determine the significance of the differences. D & E. AR and PSA immunoreactivity is present in control tumors. Cabozantinib treatment resulted in decreases in AR and PSA immunoreactivity in both models (LuCaP 23.1 (D) and C4-2B (E)). C4-2B cells express lower levels of PSA in comparison to LuCaP 23.1, and the staining is weaker in these tumor. Furthermore, large necrotic areas of tumor are present in the treated tibiae (marked by red asterics). F. 60 mg/kg cabozantinib is well tolerated up to 4 weeks in androgen-sensitive LuCaP 23.1 animals. After this period significant BW decreases vs. control were detected (up to 17%), but because of variation and number of animals, these decreases did not reach significance. 60 mg/kg cabozantinib is well tolerated up to 5 weeks in the castration-resistant C4-2B model, with a 12% significant decrease at week 6. Significance was determined by comparing enrollment BW to BW at each week using 2-sided t-test. Mean ± SEM of the groups is plotted. </p></div

A. A lower dose of cabozantinib at 30 mg/kg also inhibits tumor progression as demonstrated by decreases in serum PSA in animals bearing LuCaP 23.1 tumors in the tibiae.

<p>The lower dose was well tolerated for 6 weeks of treatment with no significant body weight loss. Prolonged treatment (7–15 weeks) caused 7–11% body weight decreases which were, however, statistically significant (P=0.0002-0.04). Significance of the changes was determined by comparing enrollment BW to BW at each week using 2-sided t-test. Mean ± SEM is plotted. B. Cabozantinib treatment (60 mg/kg) inhibited tumor growth of subcutaneous castration-resistant C4-2B tumors as determined by TV and serum PSA levels. This treatment also significantly increases survival as determined by log-rank test. C4-2B tumor growth causes decreases of BW in the experimental animals (4–13%, P= 0.0001-0.003), and the cabozantinib treatment prevents this effect resulting in no significant BW loss. Significance of the changes was determined by comparing enrollment BW to BW at each week using 2-sided t-test. Mean ± SEM of the group is plotted. </p

Cellular Adhesion Promotes Prostate Cancer Cells Escape from Dormancy

<div><p>Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of <i>in vitro</i> models. Here, we characterized <i>in vitro</i> PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant <i>in vitro</i> model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.</p></div

CDK6 overexpression induced proliferation of LuCaP PDX cells <i>in vitro</i>.

<p>LuCaP 86.2, 92 and 93 cells were lentivirally transduced to overexpress CDK6 and cultured <i>in vitro</i> to assess proliferation. Positive Ki67 indicated that CDK6 overexpression facilitated proliferation in these cells. Green, EpCAM; Red, Ki67; Blue, DAPI. Magnification: 200x. Scale bar: 20 μm.</p

Genes associated with cellular movement were downregulated in proliferating LuCaP cells.

<p>A) Heat map of hierarchically clustered differential gene expression in NG and G LuCaP PDX cells. Green, downregulated; red, upregulated. B) Ingenuity pathway analysis showing cellular movement was the top molecular and cellular function altered between NG and G cells. C) List of eight genes that were involved in the decreased activation of cellular movement in G when compared to NG cells. D) EDN1 was predicted to be the top regulator that affected the cell movement via downregulation of FN1, CDC42, and FOSL1. E) Quantitative real-time PCR showed a downregulation of FN1, CDC42 and TGFb2 in growing LuCaP lines. Data were normalized to the levels of housekeeping gene RPS15. NG: not growing; G: growing.</p

Constitutive activation of MLCK promotes proliferation of LuCaP PDX cells via upregulation of CDK6.

<p>A) LuCaP cells were infected with lentivirus containing A-tMK (constitutively activate MLCK) showed positive Ki67 staining, whereas cells transduced with an empty vector did not. B) In LuCaP 86.2, 92, and 93, ectopic expression of A-tMK induced an upregulation of CDK6 and a concurrent downregulation of E2F4 when compared to that of the empty vector-transduced cells. Inhibition of MLCK with the MLCK inhibitor ML-7 suppressed proliferation by C) abolishing Ki67 expression, D) decreasing cell viability assessed by WST-1 assay and E) downregulating CDK6 expression. E2F4 expression was not altered by the ML-7. Green, EpCAM; Red, Ki67; Blue, DAPI. Magnification: 200x. Scale bar: 20 μm. **p< 0.01 as compared to the DMSO control. CDK6: cyclin-dependent kinase 6; E2F4: E2F transcription factor 4.</p