A new murine model of osteoblastic/osteolytic lesions from human androgen-resistant prostate cancer

BACKGROUND: Up to 80% of patients dying from prostate carcinoma have developed bone metastases that are incurable. Castration is commonly used to treat prostate cancer. Although the disease initially responds to androgen blockade strategies, it often becomes castration-resistant (CRPC for Castration Resistant Prostate Cancer). Most of the murine models of mixed lesions derived from prostate cancer cells are androgen sensitive. Thus, we established a new model of CRPC (androgen receptor (AR) negative) that causes mixed lesions in bone. METHODS: PC3 and its derived new cell clone PC3c cells were directly injected into the tibiae of SCID male mice. Tumor growth was analyzed by radiography and histology. Direct effects of conditioned medium of both cell lines were tested on osteoclasts, osteoblasts and osteocytes. RESULTS: We found that PC3c cells induced mixed lesions 10 weeks after intratibial injection. In vitro, PC3c conditioned medium was able to stimulate tartrate resistant acid phosphatase (TRAP)-positive osteoclasts. Osteoprotegerin (OPG) and endothelin-1 (ET1) were highly expressed by PC3c while dikkopf-1 (DKK1) expression was decreased. Finally, PC3c highly expressed bone associated markers osteopontin (OPN), Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP) and produced mineralized matrix in vitro in osteogenic conditions. CONCLUSIONS: We have established a new CRPC cell line as a useful system for modeling human metastatic prostate cancer which presents the mixed phenotype of bone metastases that is commonly observed in prostate cancer patients with advanced disease. This model will help to understand androgen-independent mechanisms involved in the progression of prostate cancer in bone and provides a preclinical model for testing the effects of new treatments for bone metastases

Role of the orphan nuclear receptor ERRalpha in the development of breast cancer and prostate cancer bone metastases

Chez les patients atteints d’un cancer du sein ou de la prostate, le tissu osseux est souvent le siège de métastases qui présentent un phénotype essentiellement lytique dans le cas des métastases dérivant du cancer du sein, ou mixte (combinaison de lyse et de formation osseuse) dans le cas d’un cancer de la prostate. Le récepteur ERRalpha est impliqué dans la physiologie osseuse et il est considéré comme un facteur de mauvais pronostic dans ces deux types de cancers. Nous avons donc émis l’hypothèse qu'il pourrait être impliqué dans la formation des métastases osseuses associées à ces deux cancers. Nous avons observé que, dans les deux cas, ERRalpha stimule la progression tumorale au site primaire via la stimulation de l'angiogenèse et de l'expression du VEGF. Concernant les métastases osseuses dérivant du cancer du sein, ERRalpha inhibe les lésions ostéolytiques via la modulation de l'ostéoclastogenèse et de l'expression de son inhibiteur, l’OPG. A l'inverse, l'expression de ERRalpha stimule la formation de lésions lytiques induites par les cellules de cancer de la prostate en induisant l'expression du TGF-beta, de MCP-1 et de la cathépsine K, tout en induisant des zones de formation osseuse via la régulation de l'expression de l'OPG, de l’endothéline-1 et de membres de la famille Wnt. Ces résultats confirment la valeur de facteur de mauvais pronostic de ERRalpha dans la tumeur primaire. De plus, ils révèlent, pour la première fois, son implication dans le développement des métastases osseuses et suggèrent une dualité fonctionnelle de ERRalpha, comme inhibiteur et stimulateur du développement des métastases osseuses du cancer du sein et de la prostate, respectivement. Ces résultats suggèrent des mécanismes d'action différents pouvant dépendre des cellules tumorales mais aussi du microenvironnement et du statut hormonalBreast cancer and prostate cancer patients, often developed bone metastases. As ERRalpha, an orphan nuclear receptor, is involved in bone physiology and is considered as a bad prognosis factor in breast and prostate cancer, we hypothesize that it can be implicated in bone metastasis development that derived from breast and prostate cancers. While we found that, in both cases, ERRalpha stimulates the development of the primary tumor through regulation of angiogenesis and VEGF expression, we show that ERRalpha inhibits osteolytic lesions from breast cancer cells via the regulation of osteoclastogenesis and OPG expression. On the other side ERRalpha stimulates osteolytic lesions from prostate cancer through regulation of TGFbeta, MCP-1 and cathepsin K expression while inducing new bone formation combine with OPG, endothelin-1, and Wnts regulation. All together, our results confirmed ERRalpha as a bad prognosis factor in breast and prostate primary tumors. They also show a dual function of ERRalpha in bone metastasis development as an inhibitor and a stimulator of bone metastasis derived from breast and prostate cancer respectively which suggest different ERRalpha mechanisms that may depend of cancer cells but also of the microenvironment and hormonal statu

INPPL1 gene mutations in opsismodysplasia.

The INPPL1 (inositol polyphosphate phosphatase-like 1) gene encodes the inositol phosphatase, SHIP2 (for src homology 2 domain-containing inositol phosphatase 2). SHIP2 functions to dephosphorylate, and negatively regulate, the lipid second messenger phosphatidylinositol (3,4,5)P3. SHIP2 has been well studied in the area of insulin resistance and obesity but has roles in cancer and other disorders. Recently, it was reported that mutations in INPPL1 cause opsismodysplasia, a rare, autosomal recessive severe skeletal dysplasia. This review focuses on the mutations associated with opsismodysplasia and explores the role of INPPL1/ SHIP2 in skeletal development

Dual function of ERR alpha in breast cancer and bone metastasis formation: implication of VEGF and osteoprotegerin.

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRalpha) has been implicated in breast cancer and bone development, prompting us to examine whether ERRalpha may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRalpha reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRalpha upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRalpha reduced breast cancer cell growth in bone. In contrast, ERRalpha overexpression increased breast cancer cell growth in the mammary gland. ERRalpha-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRalpha mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRalpha plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone

CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

The cochaperone BCL2-associated athanogene 3 (BAG3), in complex with the heat shock protein HSPB8, facilitates mitotic rounding, spindle orientation, and proper abscission of daughter cells. BAG3 and HSPB8 mitotic functions implicate the sequestosome p62/SQSTM1, suggesting a role for protein quality control. However, the interplay between this chaperone-assisted pathway and the mitotic machinery is not known. Here, we show that BAG3 phosphorylation at the conserved T285 is regulated by CDK1 and activates its function in mitotic cell shape remodeling. BAG3 phosphorylation exhibited a high dynamic at mitotic entry and both a non-phosphorylatable BAG3T285A and a phosphomimetic BAG3T285D protein were unable to correct the mitotic defects in BAG3-depleted HeLa cells. We also demonstrate that BAG3 phosphorylation, HSPB8, and CDK1 activity modulate the molecular assembly of p62/SQSTM1 into mitotic bodies containing K63 polyubiquitinated chains. These findings suggest the existence of a mitotically regulated spatial quality control mechanism for the fidelity of cell shape remodeling in highly dividing cells

TMPRSS2:ERG gene fusion expression regulates bone markers and enhances the osteoblastic phenotype of prostate cancer bone metastases

International audienceAbstract : Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells

Expression of pro-osteoblastic factors by PC3c cells.

<p>Detection by real-time PCR of AR mRNA expression in PC3, PC3c and VCAP cancer cells lines (A), AMACR, PAP (B) and DKK1, ET-1, FGF9, Noggin, OPN, OPG, Runx2 and TGFβ mRNA expression (C and D) in PC3 and PC3c cancer cells lines. Genes expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0.05; **p<0,001, ***p<0,0001.</p

Induction of lytic and mixed bone lesions by PC3 and PC3c cells respectively after intratibial injection.

<p>(<b>A</b>) PC3 and PC3c cells were inoculated into male SCID mice; 10 weeks post inoculation, radiography revealed pure osteolytic lesions in mice injected with PC3 cells (n=6) (<b>E</b>) and mixed lesions in mice injected with PC3c cells (n=8) (<b>I</b>) compared to mice injected with PBS (n=10) (<b>A</b>) (see *(lysis) and white arrows (formation)). (<b>B,C- </b>F,G-J,K) Three-dimensional micro-CT reconstructions of tibiae and (<b>D</b>, <b>H</b>, <b>L</b>) histology after Goldner’s Trichrome staining confirmed the radiography results. T: Tumor; NB: New Bone.</p

Stimulation of osteoclasts and osteoblasts by PC3c cells <i>in vitro</i>.

<p>(<b>A</b>) Primary mouse bone marrow cells were cultured in the presence of RANKL and M-CSF and treated or not (Ct) with conditioned medium obtained from PC3 and PC3c cells. More OCs (white arrow) were formed in cultures treated with PC3c conditioned medium compared to cultures treated with PC3 conditioned medium and Ct (ANOVA, p<0.0001). (<b>B</b>) Primary mouse calvaria cell cultures were treated from day 1-21 with conditioned medium obtained from PC3 and PC3c cell. Mineralized bone nodules were present and visualized by von Kossa staining at day 21 (see mineral in black, white arrows). Mineralized bone nodule formation was decreased when primary cells were treated with conditioned medium from any of the PC3/PC3c cells (compared with non-treated (Ct) cells); the decrease was less when PC3c cell conditioned medium was used (compared with PC3) (ANOVA, p<0.001 versus Ct and versus PC3). (<b>C</b>) PC3 conditioned media stimulated the expression of OPG and RANKL in primary OBs compared with non-treated (Ct) while PC3c conditioned media only inhibits the expression of OPG compared with Ct leading to an higher RANKL/OPG ratio in PC3c conditions. (<b>D</b>) Detection by real-time PCR of SOST, DMP1, OPG and RANKL mRNA expression in MLO-Y4 cells treated with PC3 and PC3c conditioned medium. Results are plotted as the mean number of OC ± SD and OB nodules ± SD of three wells for controls and each condition and are representative of two independent experiments. Genes expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0.05; **p<0,001, ***p<0,0001.</p

PC3c cells highly expressed type I collagen.

<p>(<b>A</b>) Detection by real-time PCR of type I collagen mRNA expression in PC3c and PC3 cells cultured in normal conditions. Gene expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0,05. (<b>B</b>) Visualization of type I collagen in PC3c cells cultured on glass coverslip by electron microscopy (see black arrows).</p