Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling.

Bone morphogenetic proteins (BMPs) secreted by a variety of cell types are known to play essential roles in cell differentiation and matrix formation in the bone, cartilage, muscle, blood vessel, and neuronal tissue. BMPs activate intracellular effectors via C-terminal phosphorylation of Smad1, Smad5, and Smad9, which relay the signaling by forming a complex with Smad4 and translocate to the nucleus for transcriptional activation. Smad6 inhibits BMP signaling through diverse mechanisms operative at the membrane, cytosolic, and nuclear levels. However, the mechanistic underpinnings of Smad6 functional diversity remain unclear. Here, using a biochemical approach and cell differentiation systems, we report a cytosolic mechanism of action for Smad6 that requires arginine methylation at arginine 81 (R81) and functions through association with Smad1 and interference with the formation of Smad1-Smad4 complexes. By mutating the methylated arginine residue, R81, and by silencing the expression of protein arginine methyltransferase 1, we show that protein arginine methyltransferase 1 catalyzes R81 methylation of Smad6 upon BMP treatment, R81 methylation subsequently facilitates Smad6 interaction with the phosphorylated active Smad1, and R81 methylation facilitates Smad6-mediated interruption of Smad1-Smad4 complex formation and nuclear translocation. Furthermore, Smad6 WT but not the methylation-deficient R81A mutant inhibited BMP-responsive transcription, attenuated BMP-mediated osteogenic differentiation, and antagonized BMP-mediated inhibition of cell invasion. Taken together, our results suggest that R81 methylation plays an essential role in Smad6-mediated inhibition of BMP responses.

Hormone-Independent Sexual Dimorphism in the Regulation of Bone Resorption by Krox20.

Krox20/EGR2 is a zinc finger transcription factor, implicated in the development of the hindbrain, nerve myelination, and tumor suppression. In skeletal biology, we have demonstrated that Krox20 also regulates adult bone metabolism. We and others have characterized several functions of Krox20 in the osteoclast lineage, namely, preosteoclast proliferation and differentiation, and mature osteoclast apoptosis. We have previously reported that systemically Krox20-haploinsufficient mice have a low bone mass with increased bone resorption. However, new data have now revealed that this phenotype is restricted to females. In addition, we discovered that conditional knockout of Krox20 (cKO) restricted to osteoclast progenitors is sufficient to induce the same female-specific bone loss observed in systemic mutants. To test whether this sexual dimorphism results from an interaction between Krox20 and sex hormones, we examined the sex- and hormone-dependent role of Krox20 deficiency on proliferation and apoptosis in osteoclastic cells. Our results indicate that male and female sex hormones (dihydrotestosterone [DHT] and estradiol [E2], respectively) as well as Krox20 inhibit preosteoclast proliferation and augment osteoclast apoptosis. The observation that Krox20 expression is inhibited by DHT and E2 negates the hypothesis that the effect of sex hormones is mediated by an increase in Krox20 expression. Interestingly, the effect of Krox20 deficiency was observed only with cells derived from female animals, regardless of any sex hormones added in vitro. In addition, we have identified sexual dimorphism in the expression of several Krox20-related genes, including NAB2. This sex-specific epigenetic profile was established at puberty, maintained in the absence of sex hormones, and explains the female-specific skeletal importance of Krox20. The findings described in this study emphasize the medical importance of sex differences, which may be determined at the epigenetic level. © 2019 American Society for Bone and Mineral Research.

CLDN18.1 attenuates malignancy and related signaling pathways of lung adenocarcinoma in vivo and in vitro.

Claudins are a family of transmembrane proteins integral to the structure and function of tight junctions (TJ). Disruption of TJ and alterations in claudin expression are important features of invasive and metastatic cancer cells. Expression of CLDN18.1, the lung-specific isoform of CLDN18, is markedly decreased in lung adenocarcinoma (LuAd). Furthermore, we recently observed that aged Cldn18 -/- mice have increased propensity to develop LuAd. We now demonstrate that CLDN18.1 expression correlates inversely with promoter methylation and with LuAd patient mortality. In addition, when restored in LuAd cells that have lost expression, CLDN18.1 markedly attenuates malignant properties including xenograft tumor growth in vivo as well as cell proliferation, migration, invasion and anchorage-independent colony formation in vitro. Based on high throughput analyses of Cldn18 -/- murine lung alveolar epithelial type II cells, as well as CLDN18.1-repleted human LuAd cells, we hypothesized and subsequently confirmed by Western analysis that CLDN18.1 inhibits insulin-like growth factor-1 receptor (IGF-1R) and AKT phosphorylation. Consistent with recent data in Cldn18 -/- knockout mice, expression of CLDN18.1 in human LuAd cells also decreased expression of transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) and their target genes, contributing to its tumor suppressor activity. Moreover, analysis of LuAd cells in which YAP and/or TAZ are silenced with siRNA suggests that inhibition of TAZ, and possibly YAP, is also involved in CLDN18.1-mediated AKT inactivation. Taken together, these data indicate a tumor suppressor role for CLDN18.1 in LuAd mediated by a regulatory network that encompasses YAP/TAZ, IGF-1R and AKT signaling.

Estrogens and selective estrogen receptor modulators differentially antagonize Runx2 in ST2 mesenchymal progenitor cells.

Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1-1 nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ≥10 nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1-1 nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy.

Estrogens and androgens inhibit association of RANKL with the pre-osteoblast membrane through post-translational mechanisms.

We have recently demonstrated that RUNX2 promoted, and 17ß-Estradiol (E2) diminished, association of RANKL with the cell membrane in pre-osteoblast cultures. Here we show that, similar to E2, dihydrotestosterone (DHT) diminishes association of RANKL, and transiently transfected GFP-RANKL with the pre-osteoblast membrane without decreasing total RANKL mRNA or protein levels. Diminution of membrane-associated RANKL was accompanied with marked suppression of osteoclast differentiation from co-cultured pre-osteoclasts, even though DHT increased, not decreased, RANKL concentrations in pre-osteoblast conditioned media. A marked decrease in membrane-associated RANKL was observed after 30 min of either E2 or DHT treatment, and near-complete inhibition was observed by 1 hr, suggesting that the diminution of RANKL membrane association was mediated through non-genomic mechanisms. Further indicating dispensability of nuclear action of estrogen receptor, E2-mediated inhibition of RANKL membrane association was mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the cell nucleus. Finally, the inhibitory effect of E2 and DHT on RANKL membrane association was counteracted by the MMP inhibitor NNGH, and the effect of E2 (and not DHT) was antagonized by the Src inhibitor SU6656. Taken together, these results suggest that estrogens and androgens inhibit osteoblast-driven osteoclastogenesis through non-genomic mechanism(s) that entail, MMP-mediated RANKL dissociation from the cell membrane.

Relationship between RUNX1 and AXIN1 in ER-negative versus ER-positive Breast Cancer.

RUNX1 plays opposing roles in breast cancer: a tumor suppressor in estrogen receptor-positive (ER+) disease and an oncogenic role in ER-negative (ER-) tumors. Potentially mediating the former, we have recently reported that RUNX1 prevents estrogen-driven suppression of the mRNA encoding the tumor suppressor AXIN1. Accordingly, AXIN1 protein expression was diminished upon RUNX1 silencing in ER+ breast cancer cells and was positively correlated with AXIN1 protein expression across tumors with high levels of ER. Here we report the surprising observation that RUNX1 and AXIN1 proteins are strongly correlated in ER- tumors as well. However, this correlation is not attributable to regulation of AXIN1 by RUNX1 or vice versa. The unexpected correlation between RUNX1, playing an oncogenic role in ER- breast cancer, and AXIN1, a well-established tumor suppressor hub, may be related to a high ratio between the expression of variant 2 and variant 1 (v2/v1) of AXIN1 in ER- compared with ER+ breast cancer. Although both isoforms are similarly regulated by RUNX1 in estrogen-stimulated ER+ breast cancer cells, the higher v2/v1 ratio in ER- disease is expected to weaken the tumor suppressor activity of AXIN1 in these tumors.

Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation.

Inhibition of Runx2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)-induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2(dox) cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2-driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT-qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co-treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox-driven alkaline phosphatase (ALP) activity in control ST2/Rx2(dox) cells, but not in cells in which Wif1 was silenced. Unlike its anti-mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S-phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2-driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2(dox) cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. J. Cell. Physiol. 232: 145-153, 2017. © 2016 Wiley Periodicals, Inc.

High-Throughput Screen for Inhibitors of Androgen Receptor-RUNX2 Transcriptional Regulation in Prostate Cancer.

Runt-related transcription factor 2 (RUNX2) plays a critical role in prostate cancer progression. RUNX2 interacts with the androgen receptor (AR) and modulates its transcriptional activity in a locus-specific manner. RUNX2 and AR synergistically stimulate a subset of genes, including the pro-oncogene snail family zinc finger 2 (SNAI2). AR-RUNX2 signaling cooperatively induces invasiveness of prostate cancer cells via SNAI2; and coexpression of AR, RUNX2, and SNAI2 in prostate cancer biopsy samples predicts disease recurrence. Competitive inhibition of AR alone could not disrupt the synergistic activation of SNAI2. We therefore established a phenotypic cell-based screening assay for compounds that could inhibit AR-RUNX2 synergistic activity either directly or indirectly. This assay was used to screen 880 compounds as a proof of concept, resulting in identification of several compounds that disrupted the synergistic stimulation of genes. Further investigation suggested the involvement of epidermal growth factor receptor (EGFR) signaling in AR/RUNX2 synergistic activity. Our assay is amenable to high-throughput screening and can be used to identify inhibitors of the AR-RUNX2 interaction in prostate cancer cells.

Autophagic UVRAG Promotes UV-Induced Photolesion Repair by Activation of the CRL4(DDB2) E3 Ligase.

UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.

RUNX1 prevents oestrogen-mediated AXIN1 suppression and ß-catenin activation in ER-positive breast cancer.

Recent high-throughput studies revealed recurrent RUNX1 mutations in breast cancer, specifically in oestrogen receptor-positive (ER(+)) tumours. However, mechanisms underlying the implied RUNX1-mediated tumour suppression remain elusive. Here, by depleting mammary epithelial cells of RUNX1 in vivo and in vitro, we demonstrate combinatorial regulation of AXIN1 by RUNX1 and oestrogen. RUNX1 and ER occupy adjacent elements in AXIN1's second intron, and RUNX1 antagonizes oestrogen-mediated AXIN1 suppression. Accordingly, RNA-seq and immunohistochemical analyses demonstrate an ER-dependent correlation between RUNX1 and AXIN1 in tumour biopsies. RUNX1 loss in ER(+) mammary epithelial cells increases ß-catenin, deregulates mitosis and stimulates cell proliferation and expression of stem cell markers. However, it does not stimulate LEF/TCF, c-Myc or CCND1, and it does not accelerate G1/S cell cycle phase transition. Finally, RUNX1 loss-mediated deregulation of ß-catenin and mitosis is ameliorated by AXIN1 stabilization in vitro, highlighting AXIN1 as a potential target for the management of ER(+) breast cancer.

Glucocorticoid-Induced Osteoporosis.

Osteoporosis is among the most devastating side effects of glucocorticoid (GC) therapy for the management of inflammatory and auto-immune diseases. Evidence from both humans and mice indicate deleterious skeletal effects within weeks of pharmacological GC administration, both related and unrelated to a decrease in bone mineral density (BMD). Osteoclast numbers and bone resorption are also rapidly increased, and together with osteoblast inactivation and decreased bone formation, these changes lead the fastest loss in BMD during the initial disease phase. Bone resorption then decreases to sub-physiological levels, but persistent and severe inhibition of bone formation leads to further bone loss and progressively increased fracture risk, up to an order of magnitude higher than that observed in untreated individuals. Bone forming osteoblasts are thus considered the main culprits in GC-induced osteoporosis (GIO). Accordingly, we focus this review primarily on deleterious effects on osteoblasts: inhibition of cell replication and function and acceleration of apoptosis. Mediating these adverse effects, GCs target pivotal regulatory mechanisms that govern osteoblast growth, differentiation and survival. Specifically, GCs inhibit growth factor pathways, including Insulin Growth Factors, Growth Hormone, Hepatocyte Growth/Scatter Factor and IL6-type cytokines. They also inhibit downstream kinases, including PI3-kinase and the MAP kinase ERK, the latter attributable in part to direct transcriptional stimulation of MAP kinase phosphatase 1. Most importantly, however, GCs inhibit the Wnt signaling pathway, which plays a pivotal role in osteoblast replication, function and survival. They transcriptionally stimulate expression of Wnt inhibitors of both the Dkk and Sfrp families, and they induce reactive oxygen species (ROS), which result in loss of ß-catenin to ROS-activated FoxO transcription factors. Identification of dissociated GCs, which would suppress the immune system without causing osteoporosis, is proving more challenging than initially thought, and GIO is currently managed by co-treatment with bisphosphonates or PTH. These drugs, however, are not ideally suited for GIO. Future therapeutic approaches may aim at GC targets such as those mentioned above, or newly identified targets including the Notch pathway, the AP-1/Il11 axis and the osteoblast master regulator RUNX2.

Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis.

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of BM-derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)-4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven toward osteoblastic differentiation revealed an increase in vascular endothelial growth factor A (Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC-derived VEGFA mRNA and protein and that this was enhanced by BMP-4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA- mediated knock-down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP-4-treated cocultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma-derived intracrine VEGFA in osteoblastogenesis.

Estrogens antagonize RUNX2-mediated osteoblast-driven osteoclastogenesis through regulating RANKL membrane association.

In addition to its thoroughly investigated role in bone formation, the osteoblast master transcription factor RUNX2 also promotes osteoclastogenesis and bone resorption. Here we demonstrate that 17ß-estradiol (E2), strongly inhibits RUNX2-mediated osteoblast-driven osteoclastogenesis in co-cultures. Towards deciphering the underlying mechanism, we induced premature expression of RUNX2 in primary murine pre-osteoblasts, which resulted in robust differentiation of co-cultured splenocytes into mature osteoclasts. This was attributable to RUNX2-mediated increase in RANKL secretion, determined by ELISA, as well as to RUNX2-mediated increase in RANKL association with the osteoblast membrane, demonstrated using confocal fluorescence microscopy. The increased association with the osteoblast membrane was recapitulated by transiently expressed GFP-RANKL. E2 abolished the RUNX2-mediated increase in membrane-associated RANKL and GFP-RANKL, as well as the concomitant osteoclastogenesis. RUNX2-mediated RANKL cellular redistribution was attributable in part to a decrease in Opg expression, but E2 did not influence Opg expression either in the presence or absence of RUNX2. Diminution of RUNX2-mediated osteoclastogenesis by E2 occurred regardless of whether the pre-osteoclasts were derived from wild type or estrogen receptor alpha (ERα)-knockout mice, suggesting that activated ERα inhibited osteoblast-driven osteoclastogenesis by acting in osteoblasts, possibly targeting RUNX2. Indeed, microarray analysis demonstrated global attenuation of the RUNX2 response by E2, including abrogation of Pstpip2 expression, which likely plays a critical role in membrane trafficking. Finally, the selective ER modulators (SERMs) tamoxifen and raloxifene mimicked E2 in abrogating the stimulatory effect of osteoblastic RUNX2 on osteoclast differentiation in the co-culture assay. Thus, E2 antagonizes RUNX2-mediated RANKL trafficking and subsequent osteoclastogenesis. Targeting RUNX2 and/or downstream mechanisms that regulate RANKL trafficking may lead to the development of improved SERMs and possibly non-hormonal therapeutic approaches to high turnover bone disease.

Initial Characterization of Osteoblast Differentiation and Loss of RUNX2 Stability in the Newly Established SK11 Human Embryonic Stem Cell-Derived Cell Line.

We describe a novel model for investigation of genetically normal human osteoblasts in culture. SK11 is a clonal progenitor cell line derived from human embryonic stem cells. Initially selected based on the expression of chondrogenic markers when differentiated in micromass culture, SK11 cells display typical mRNA expression patterns of bone phenotypic genes under osteogenic conditions. These include osterix, α1(I) collagen, alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. Similar to well-characterized murine osteoblast cultures, the osteoblast master regulator RUNX2 was present during the first few days after plating, but the protein disappeared during the first week of culture. Loss of RUNX2 expression is considered an important regulatory feature for osteoblast maturation. Indeed, following ∼2 weeks of differentiation, SK11 cultures exhibited robust calcium deposition, evidenced by alizarin red staining. We also introduced a lentiviral vector encoding doxycycline (dox)-inducible FLAG-tagged RUNX2 into SK11 cells. Dox-mediated enhancement of RUNX2 expression resulted in accelerated mineralization, which was further increased by co-treatment with BMP-2. Like the endogenous RUNX2, expression of the virally coded FLAG-RUNX2 was lost during the first week of culture despite persistent dox treatment. By following RUNX2 decay after dox withdrawal from day-5 versus day-3 cultures, we demonstrated a developmentally regulated decrease in RUNX2 stability. Availability of culture models for molecular investigation of genetically normal human osteoblasts is important because differences between murine and human osteoblasts, demonstrated here by the regulation of matrix Gla Protein, may have significant biomedical implications.

Dependence of castration-resistant prostate cancer (CRPC) stem cells on CRPC-associated fibroblasts.

We previously established a role for cancer-associated fibroblasts (CAF) in enhancing the self-renewal and differentiation potentials of putative prostate cancer stem cells (CSC). Our published work focused on androgen-dependent prostate cancer (ADPC) using the conditional Pten deletion mouse model. Employing the same model, we now describe the interaction of CAF and CSC in castration-resistant prostate cancer (CRPC). CAF isolated from ADPC (ADPCAF) and from CRPC (CRPCAF) were compared in terms of their ability to support organoid formation and tumor initiation by CSC from CRPC (CRPCSC) in vitro and in vivo. CRPCSC formed spheroids in vitro and well-differentiated glandular structures under the renal capsules of recipient mice in vivo more effectively in the presence of CRPCAF compared to ADPCAF. Furthermore, whereas CSC with CAF from ADPC formed mostly well-differentiated tumors in our previous study, we now show that CRPCSC, when combined with CRPCAF (but not ADPCAF), can form aggressive, poorly-differentiated tumors. The potential of CRPCAF to support organoid/tumor formation by CRPCSC remained greater even when compared to 10-fold more ADPCAF, suggesting that paracrine factors produced specifically by CRPCAF preferentially potentiate the stemness and tumorigenic properties of the corresponding CSC. This apparently unique property of CRPCAF was notable when the CAF and CSC were grafted in either intact or castrated recipient mice. In both environments, CRPCAF induced in the epithelial compartment higher proliferative activity compared to ADPCAF, indicated by a higher Ki67 index. Factors released by CRPCAF to regulate CRPCSC may be targeted to develop novel therapeutic approaches to manage advanced prostate cancer.

Runx1-mediated regulation of osteoclast differentiation and function.

Excessive bone resorption is the cause of several metabolic bone diseases including osteoporosis. Thus, identifying factors that can inhibit osteoclast formation and/or activity may define new drug targets that can be used to develop novel therapies for these conditions. Emerging evidence demonstrates that the master regulator of hematopoiesis, Runx1, is expressed in preosteoclasts and may influence skeletal health. To examine the potential role of Runx1 in osteoclast formation and function, we deleted its expression in myeloid osteoclast precursors by crossing Runx1 floxed mice (Runx1(F/F)) with CD11b-Cre transgenic mice. Mice lacking Runx1 in preosteoclasts (CD11b-Cre;Runx1(F/F)) exhibited significant loss of femoral trabecular and cortical bone mass compared with that in Cre-negative mice. In addition, serum levels of collagen type 1 cross-linked C-telopeptide, a biomarker of osteoclast-mediated bone resorption, were significantly elevated in CD11b-Cre;Runx1(F/F) mice compared with those in Runx1(F/F) mice. Tartrate-resistant acid phosphatase-positive osteoclasts that differentiated from bone marrow cells of CD11b-Cre;Runx1(F/F) mice in vitro were larger, were found in greater numbers, and had increased bone resorbing activity than similarly cultured cells from Runx1(F/F) mice. CD11b-Cre;Runx1(F/F) bone marrow cells that were differentiated into osteoclasts in vitro also had elevated mRNA levels of osteoclast-related genes including vacuolar ATPase D2, cathepsin K, matrix metalloproteinase 9, calcitonin receptor, osteoclast-associated receptor, nuclear factor of activated T cells cytoplasmic 1, and cFos. These data indicate that Runx1 expression in preosteoclasts negatively regulates osteoclast formation and activity and contributes to overall bone mass.

Differential effects of RUNX2 on the androgen receptor in prostate cancer: synergistic stimulation of a gene set exemplified by SNAI2 and subsequent invasiveness.

Changes to androgen signaling during prostate carcinogenesis are associated with both inhibition of cellular differentiation and promotion of malignant phenotypes. The androgen receptor (AR)-binding transcription factor RUNX2 has been linked to prostate cancer progression but the underlying mechanisms have not been fully defined. In this study, we investigated the genome-wide influence of RUNX2 on androgen-induced gene expression and AR DNA binding in prostate cancer cells. RUNX2 inhibited the androgen response partly by promoting the dissociation of AR from its target genes such as the tumor suppressor NKX3-1. However, AR activity persists in the presence of RUNX2 at other AR target genes, some of which are cooperatively stimulated by androgen and RUNX2 signaling. These genes are associated with putative enhancers co-occupied by AR and RUNX2. One such gene, the invasion-promoting Snail family transcription factor SNAI2, was co-activated by AR and RUNX2. Indeed, these two transcription factors together, but neither alone stimulated prostate cancer cell invasiveness, which could be abolished by SNAI2 silencing. Furthermore, an immunohistochemical analysis of SNAI2 in archived primary prostate cancer specimens revealed a correlation with the RUNX2 histoscore, and simultaneous strong staining for SNAI2, RUNX2, and AR (but not any pair alone) was associated with disease recurrence. Overall, our findings suggest cooperation between AR and RUNX in the stimulation of oncogenes such as SNAI2, which might be targeted for individualized prostate cancer therapy.

Glucocorticoids antagonize RUNX2 during osteoblast differentiation in cultures of ST2 pluripotent mesenchymal cells.

The efficacy of glucocorticoids (GCs) in treating a wide range of autoimmune and inflammatory conditions is blemished by severe side effects, including osteoporosis. The chief mechanism leading to GC-induced osteoporosis is inhibition of bone formation, but the role of RUNX2, a master regulator of osteoblast differentiation and bone formation, has not been well studied. We assessed effects of the synthetic GC dexamethasone (dex) on transcription of RUNX2-stimulated genes during the differentiation of mesenchymal pluripotent cells into osteoblasts. Dex inhibited a RUNX2 reporter gene and attenuated locus-dependently RUNX2-driven expression of several endogenous target genes. The anti-RUNX2 activity of dex was not attributable to decreased RUNX2 expression, but rather to physical interaction between RUNX2 and the GC receptor (GR), demonstrated by co-immunoprecipitation assays and co-immunofluorescence imaging. Investigation of the RUNX2/GR interaction may lead to the development of bone-sparing GC treatment modalities for the management of autoimmune and inflammatory diseases.

Prolactin-induced protein (PIP) regulates proliferation of luminal A type breast cancer cells in an estrogen-independent manner.

Prolactin-induced Protein (PIP), an aspartyl protease unessential for normal mammalian cell function, is required for the proliferation and invasion of some breast cancer (BCa) cell types. Because PIP expression is particularly high in the Luminal A BCa subtype, we investigated the roles of PIP in the related T47D BCa cell line. Nucleic acid and antibody arrays were employed to screen effects of PIP silencing on global gene expression and activation of receptor tyrosine kinases (RTKs), respectively. Expression of PIP-stimulated genes, as defined in the T47D cell culture model, was well correlated with the expression of PIP itself across a cohort of 557 mRNA profiles of diverse BCa tumors, and bioinformatics analysis revealed cJUN and cMYC as major nodes in the PIP-dependent gene network. Among 71 RTKs tested, PIP silencing resulted in decreased phosphorylation of focal adhesion kinase (FAK), ephrin B3 (EphB3), FYN, and hemopoietic cell kinase (HCK). Ablation of PIP also abrogated serum-induced activation of the downstream serine/threonine kinases AKT, ERK1/2, and JNK1. Consistent with these results, PIP-depleted cells exhibited defects in adhesion to fibronectin, cytoskeletal stress fiber assembly and protein secretion. In addition, PIP silencing abrogated the mitogenic response of T47D BCa cells to estradiol (E2). The dependence of BCa cell proliferation was unrelated, however, to estrogen signaling because: 1) PIP silencing did not affect the transcriptional response of estrogen target genes to hormone treatment, and 2) PIP was required for the proliferation of tamoxifen-resistant BCa cells. Pharmacological inhibition of PIP may therefore serve the bases for both augmentation of existing therapies for hormone-dependent tumors and the development of novel therapeutic approaches for hormone-resistant BCa.

Contextual effect of repression of bone morphogenetic protein activity in prostate cancer.

Several studies have focused on the effect of bone morphogenetic protein (BMP) on prostate cancer homing and growth at distant metastatic sites, but very little effect at the primary site. Here, we used two cell lines, one (E8) isolated from a primary tumor and the other (cE1) from a recurrent tumor arising at the primary site, both from the conditional Pten deletion mouse model of prostatic adenocarcinoma. Over-expression of the BMP antagonist noggin inhibited proliferation of cE1 cells in vitro while enhancing their ability to migrate. On the other hand, cE1/noggin grafts grown in vivo showed a greater mass and a higher proliferation index than the cE1/control grafts. For suppression of BMP activity in the context of cancer-associated fibroblasts (CAFs), we used noggin-transduced CAFs from the same mouse model to determine their effect on E8- or cE1-induced tumor growth. CAF/noggin led to increased tumor mass and greater de-differentiation of the E8 cell when compared with tumors formed in the presence of CAF/control cells. A trend of increase in the size of the tumor was also noted for cE1 cells when inoculated with CAF/noggin. Together, the results may point to a potential inhibitory role of BMP in the growth or re-growth of prostate tumor at the primary site. Additionally, results for cE1/noggin, and cE1 mixed with CAF/noggin, suggested that suppression of BMP activity in the cancer cells may have a stronger growth-enhancing effect on the tumor than its suppression in the fibroblastic compartment of the tumor microenvironment.