Functional biology of the Steel syndrome founder allele and evidence for clan genomics derivation of COL27A1 pathogenic alleles worldwide.

Previously we reported the identification of a homozygous COL27A1 (c.2089G>C; p.Gly697Arg) missense variant and proposed it as a founder allele in Puerto Rico segregating with Steel syndrome (STLS, MIM #615155); a rare osteochondrodysplasia characterized by short stature, congenital bilateral hip dysplasia, carpal coalitions, and scoliosis. We now report segregation of this variant in five probands from the initial clinical report defining the syndrome and an additional family of Puerto Rican descent with multiple affected adult individuals. We modeled the orthologous variant in murine Col27a1 and found it recapitulates some of the major Steel syndrome associated skeletal features including reduced body length, scoliosis, and a more rounded skull shape. Characterization of the in vivo murine model shows abnormal collagen deposition in the extracellular matrix and disorganization of the proliferative zone of the growth plate. We report additional COL27A1 pathogenic variant alleles identified in unrelated consanguineous Turkish kindreds suggesting Clan Genomics and identity-by-descent homozygosity contributing to disease in this population. The hypothesis that carrier states for this autosomal recessive osteochondrodysplasia may contribute to common complex traits is further explored in a large clinical population cohort. Our findings augment our understanding of COL27A1 biology and its role in skeletal development; and expand the functional allelic architecture in this gene underlying both rare and common disease phenotypes.

CXCR2: A Novel Mediator of Mammary Tumor Bone Metastasis.

Most breast cancer patients die due to bone metastasis. Although metastasis accounts for 5% of the breast cancer cases, it is responsible for most of the deaths. Sometimes even before the detection of a primary tumor, most of the patients have bone and lymph node metastasis. Moreover, at the time of death, breast cancer patients have the bulk of the tumor burden in their bones. Therapy options are available for the treatment of primary tumors, but there are minimal options for treating breast cancer patients who have bone metastasis. C-X-C motif chemokine receptor type 2 (CXCR2) receptor-mediated signaling has been shown to play a critical role during bone-related inflammations and its ligands C-X-C motif chemokine ligand 6 (CXCL6) and 8 (CXCL8) aid in the resorption of bone during bone metastasis. In this study, we tested the hypothesis that CXCR2 contributes to mammary tumor-induced osteolysis and bone metastasis. In the present study, we examined the role of both tumor cell-derived and host-derived CXCR2 in influencing mammary tumor cell bone metastasis. For understanding the role of tumor cell-derived CXCR2, we utilized Cl66 CXCR2 knockdown (Cl66-shCXCR2) and Cl66-Control cells (Cl66-Control) and observed a significant decrease in tumor growth and tumor-induced osteolysis in Cl66-shCXCR2 cells in comparison with the Cl66-Control cells. Next, for understanding the role of host-derived CXCR2, we utilized mice with genomic knockdown of CXCR2 (Cxcr2-/-) and injected Cl66-Luciferase (Cl66-Luc) or 4T1-Luciferase (4T1-Luc) cells. We observed decreased bone destruction and metastasis in the bone of Cxcr2-/- mice. Our data suggest the importance of both tumor cell- and host-derived CXCR2 signaling in the bone metastasis of breast cancer cells.

Host Cxcr2-dependent regulation of mammary tumor growth and metastasis.

Host-derived angiogenic and inflammatory tumor supportive microenvironment regulates progression and metastasis, but the molecular mechanism(s) underlying host-tumor interactions remains unclear. Tumor expression of CXCR2 and its ligands have been shown to regulate angiogenesis, invasion, tumor growth, and metastasis. In this report, we hypothesized that host-derived Cxcr2-dependent signaling plays an important role in breast cancer growth and metastasis. Two mammary tumor cell lines Cl66 and 4T1 cells were orthotopically implanted into the mammary fat pad of wild-type and Cxcr2(-/-) female BALB/c mice. Tumor growth and spontaneous lung metastasis were monitored. Immunohistochemical analyses of the tumor tissues were performed to analyze proliferation, angiogenesis, apoptosis and immune cell infiltration. Our results demonstrated that knock-down of host Cxcr2 decreases tumor growth and metastasis by reducing angiogenesis, proliferation and enhancing apoptosis. Host Cxcr2 plays an important role in governing the pro-inflammatory response in mammary tumors as evaluated by decreased Gr1(+) tumor-associated granulocytes, F4/80(+) tumor associated macrophages, and CD11b(+)Gr1(+) myeloid derived suppressor cells in Cxcr2(-/-) mice as compared to control wild-type mice. Together, these results demonstrate that host Cxcr2-dependent signaling regulates mammary tumor growth and metastasis by promoting angiogenesis and pro-inflammatory responses.

Targeting CXCR2 enhances chemotherapeutic response, inhibits mammary tumor growth, angiogenesis, and lung metastasis.

Breast cancer is one of the leading causes of cancer deaths among females. Many challenges exist in the current management of advanced stage breast cancer as there are fewer recognized therapeutic strategies, often because of therapy resistance. How breast cancer cells evade chemotherapy and the underlying mechanism remains unclear. We and others have observed that malignant cells that survive initial chemo- and radiation therapy express higher levels of CXCR2 ligands, which may provide a survival benefit leading to therapy resistance. In this report, we test the hypothesis that CXCR2-dependent signaling in malignant cells may be critical for chemotherapy resistance and targeting this signaling axis may enhance the antitumor and antimetastatic activity of chemotherapeutic drugs and limit their toxicity. We used Cl66-wt, 4T1-wt, Cl66sh-CXCR2, and 4T1sh-CXCR2 cells expressing differential levels of the CXCR2 receptor to evaluate the role of targeting CXCR2 on chemotherapeutic responses. Knockdown of CXCR2 enhances paclitaxel and doxorubicin-mediated toxicity at suboptimal doses. Moreover, we observed an increase in the expression of CXCL1, a CXCR2 ligand in paclitaxel and doxorubicin-treated mammary tumor cells, which were inhibited following CXCR2 knockdown. Knockdown of CXCR2 enhanced antitumor activity of paclitaxel in an in vivo mammary tumor model. We observed significant inhibition of spontaneous lung metastases in animals bearing CXCR2 knockdown tumors and treated with paclitaxel as compared with the control group. Our data suggest the novel role of CXCR2 and its ligands in maintaining chemotherapy resistance and provide evidence that targeting CXCR2 signaling in an adjuvant setting will help circumvent chemotherapy resistance.

Role of chemokine receptor CXCR2 expression in mammary tumor growth, angiogenesis and metastasis.

BACKGROUND: Chemokines and their receptors have long been known to regulate metastasis in various cancers. Previous studies have shown that CXCR2 expression is upregulated in malignant breast cancer tissues but not in benign ductal epithelial samples. The functional role of CXCR2 in the metastatic phenotype of breast cancer still remains unclear. We hypothesize that the chemokine receptor, CXCR2, mediates tumor cell invasion and migration and promotes metastasis in breast cancer. The objective of this study is to investigate the potential role of CXCR2 in the metastatic phenotype of mouse mammary tumor cells. MATERIALS AND METHODS: We evaluated the functional role of CXCR2 in breast cancer by stably downregulating the expression of CXCR2 in metastatic mammary tumor cell lines Cl66 and 4T1, using short hairpin RNA (shRNA). The effects of CXCR2 downregulation on tumor growth, invasion and metastatic potential were analyzed in vitro and in vivo. RESULTS: We demonstrated knock down of CXCR2 in Cl66 and 4T1 cells (Cl66-shCXCR2 and 4T1-shCXCR2) cells by reverse transcriptase polymerase chain reaction (RT-PCR) at the transcriptional level and by immunohistochemistry at the protein level. We did not observe a significant difference in in vitro cell proliferation between vector control and CXCR2 knock-down Cl66 or 4T1 cells. Next, we examined the invasive potential of Cl66-shCXCR2 cells by in vitro Matrigel invasion assay. We observed a significantly lower number (52 ± 5) of Cl66-shCXCR2 cells invading through Matrigel compared to control cells (Cl66-control) (182 ± 3) (P < 0.05). We analyzed the in vivo metastatic potential of Cl66-shCXCR2 using a spontaneous metastasis model by orthotopically implanting cells into the mammary fat pad of female BALB/c mice. Animals were sacrificed 12 weeks post tumor implantation and tissue samples were analyzed for metastatic nodules. CXCR2 downregulation significantly inhibited tumor cell metastasis. All the mice (n = 10) implanted with control Cl66 cells spontaneously developed lung metastasis, whereas a significantly lower number of mice (40%) implanted with Cl66-shCXCR2 cells exhibited lung metastases. CONCLUSIONS: Together, these results suggest that CXCR2 may play a critical role in breast cancer invasion and metastasis.

Tumor-stromal interactions in bone metastasis.

The metastasis of tumor cells to distant organs is the primary cause of cancer-related mortality in most cancers. The interaction of tumor cells with local stroma at the metastatic site plays a critical role in metastatic dissemination and the establishment of metastases. These tumor-stromal interactions regulate several important steps including degradation of extracellular matrix, release of sequestered growth factors, and expression of chemokines, cytokines, and receptors on tumor cells and the interacting stromal cells. Breast, prostate, and lung cancers preferentially metastasize to bone. Tumor cell interactions with the bone microenvironment initiate a series of complex cellular interactions that promotes establishment of osteoclastic and/or osteoblastic metastasis. Understanding the interactions between tumor cells and the stroma is important to identify molecular targets to develop novel therapies aimed at reducing metastasis formation. In this article, we review the important mechanisms of tumor-stromal interaction in the development of bone metastasis.

Matrix metalloproteinase (MMP)-13 regulates mammary tumor-induced osteolysis by activating MMP9 and transforming growth factor-beta signaling at the tumor-bone interface.

The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-kappaB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-beta signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis.

Small interfering RNA-mediated CXCR1 or CXCR2 knock-down inhibits melanoma tumor growth and invasion.

CXCR1 and CXCR2 are receptors for CXCL-8 and are differentially expressed on melanoma and endothelial cells. In this study, we determined the functional role of these receptors in melanoma progression. We stably knock-down the expression of CXCR1 and/or CXCR2 in A375-SM (SM; high metastatic) human melanoma cells by short-hairpin RNA transfection. Cell proliferation, migration, invasion, ERK phosphorlyation and cytoskeletal rearrangements were carried out in vitro. In vivo growth was evaluated using murine subcutaneous xenograft model. Our data demonstrate that knock-down of CXCR1 and/or CXCR2 expression, inhibited melanoma cell proliferation, survival, migration and invasive potential in vitro. Moreover, we also observed inhibition of ERK phosphorylation and cytoskeltal rearrangement in SM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2 cells. Furthermore, when SM-shCXCR1 or SM-shCXCR2 cells implanted in nude mice, tumor growth, proliferation and microvessel density was significantly inhibited as compared to SM-control cells. In addition, we observed a significant increase in melanoma cell apoptosis in SM-shCXCR1 and SM-shCXCR2 tumors compared to SM-control tumors. Together, these data demonstrate that CXCR1 and CXCR2 expression play a critical role in human melanoma tumor progression and, functional blockade of CXCR1 and CXCR2 could be potentially used for future therapeutic intervention in malignant melanoma.

Cathepsin G-mediated enhanced TGF-beta signaling promotes angiogenesis via upregulation of VEGF and MCP-1.

Transforming growth factor (TGF)-beta signaling makes a significant contribution to the pathogenesis of breast cancer bone metastasis. In other tumor types, TGF-beta has been shown to promote tumor vascularity. Here, we report that inhibition of TGF-beta significantly reduces microvessel density in mammary tumor-induced bone lesions, mediated by decreased expression of both vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1, both known angiogenic factors. Cathepsin G upregulation at the tumor-bone interface has been linked to increased TGF-beta signaling, and we also report that inhibition of Cathepsin G reduced tumor vascularity, as well as VEGF and MCP-1 expression.

Cathepsin G-mediated activation of pro-matrix metalloproteinase 9 at the tumor-bone interface promotes transforming growth factor-beta signaling and bone destruction.

Increased transforming growth factor-beta (TGF-beta) signaling has been observed at the tumor-bone interface of mammary tumor-induced osteolytic lesions despite no observed transcriptional up-regulation of TGF-beta. To this point, the mechanism for enhanced TGF-beta signaling remains unclear. The bulk of TGF-beta that is released at the tumor-bone interface is in an inactive form secondary to association with beta-latency-associated protein and latency TGF-beta binding protein. We hypothesized that the observed increase in TGF-beta signaling is due to increased cathepsin G-dependent, matrix metalloproteinase 9 (MMP9)-mediated activation of latent TGF-beta. MMP9 is capable of activating latent TGF-beta, and we observed that decreased production of MMP9 was associated with reduced TGF-beta signaling. Similar to TGF-beta, MMP9 is released in an inactive form and requires proteolytic activation. We showed that cathepsin G, which we have previously shown to be up-regulated at the tumor-bone interface, is capable of activating pro-MMP9. Inhibition of cathepsin G in vivo significantly reduced MMP9 activity, increased the ratio of latent TGF-beta to active TGF-beta, and reduced the level of TGF-beta signaling. Our proposed model based on these results is that cathepsin G is up-regulated through tumor-stromal interactions and activates pro-MMP9, active MMP9 cleaves and releases active TGF-beta, and active TGF-beta can then promote tumor growth and enhance osteoclast activation and subsequent bone resorption. Thus, for the first time, we have identified cathepsin G and MMP9 as proteases involved in enhanced TGF-beta signaling at the tumor-bone interface of mammary tumor-induced osteolytic lesions and have identified these proteases as potential therapeutic targets.

Enhanced expression and shedding of receptor activator of NF-kappaB ligand during tumor-bone interaction potentiates mammary tumor-induced osteolysis.

The bone microenvironment plays a critical role in tumor-induced osteolysis and osteolytic metastasis through tumor-bone (TB)-interaction. Receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) is one of the critical signaling molecules involved in osteolysis and bone metastasis. However, the regulation and functional significance of RANKL at the TB-interface in tumor-induced osteolysis remains unclear. In this report, we examined the role of tumor-stromal interaction in the regulation of RANKL expression and its functional significance in tumor-induced osteolysis. Using a novel mammary tumor model, we identified that RANKL expression was upregulated at the TB-interface as compared to the tumor alone area. We demonstrate increased generation of sRANKL at the TB-interface, which is associated with tumor-induced osteolysis. The ratio of RANKL to osteoprotegrin (OPG), a decoy receptor for RANKL, at the TB-interface was also increased. Targeting RANKL expression with antisense oligonucleotides (RANKL-ASO), significantly abrogated tumor-induced osteolysis, decreased RANKL expression and the RANKL:OPG ratio at the TB-interface. Together, these results demonstrate that upregulation of RANKL expression and sRANKL generation at the TB-interface potentiates tumor-induced osteolysis.

Cathepsin G recruits osteoclast precursors via proteolytic activation of protease-activated receptor-1.

Metastatic breast cancer shows extreme tropism for the bone microenvironment, leading to the establishment of osteolytic metastases. Perpetuation of tumor-induced osteolysis requires a continuous supply of osteoclast precursors migrating into the bone microenvironment that can subsequently differentiate into mature osteoclasts and resorb bone. Thus, identification and subsequent targeting of chemoattractants of osteoclast precursors that are up-regulated at the tumor-bone interface represents a potential avenue to interrupt osteolysis. We report that cathepsin G, a serine protease, plays a vital role in the bone microenvironment by modulating tumor-stromal interaction in a manner that favors tumor establishment and regulates chemotaxis of monocytes, a subset of which has the potential to differentiate into osteoclasts. Our data show that cathepsin G-induced chemotaxis of monocytes is mediated by proteolytic activation of protease-activated receptor-1 (PAR-1). Attenuation of PAR-1 activation abrogates cathepsin G-mediated induction of monocyte chemotaxis. We also show that in vivo inhibition of cathepsin G reduces the number of CD11b(+) osteoclast precursors and mature osteoclasts at the tumor-bone interface. Together, these data suggest that therapeutic targeting of both PAR-1 signaling in osteoclast precursors as well as cathepsin G at the tumor-bone interface has the potential to reduce osteolysis by inhibiting the recruitment, differentiation, and activation of osteoclast precursors.

Small-molecule antagonists for CXCR2 and CXCR1 inhibit human melanoma growth by decreasing tumor cell proliferation, survival, and angiogenesis.

PURPOSE: Melanoma, the most aggressive form of skin cancer, accounts for 75% of all skin cancer-related deaths and current therapeutic strategies are not effective in advanced disease. In the current study, we have investigated the efficacy of orally active small-molecule antagonist targeting CXCR2/CXCR1. EXPERIMENTAL DESIGN: Human A375SM melanoma cells were treated with SCH-479833 or SCH-527123, and their effect on proliferation, motility, and invasion was evaluated in vitro. We examined the downstream signaling events in the cells following treatment with antagonists. For in vivo studies, A375SM cells were implanted subcutaneously into athymic nude mice followed by administration of SCH-479833, SCH-527123, or hydroxypropyl-beta-cyclodextrin (20%) orally for 21 days and their effect on tumor growth and angiogenesis was evaluated. RESULTS: Our data show that SCH-479833 or SCH-527123 inhibited the melanoma cell proliferation, chemotaxis, and invasive potential in vitro. Treatment of melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth. Histologic and histochemical analyses showed significant (P < 0.05) decreases in tumor cell proliferation and microvessel density in tumors. Moreover, we observed a significant increase in melanoma cell apoptosis in SCH-479833- or SCH-527123-treated animals compared with controls. CONCLUSION: Together, these studies show that selectively targeting CXCR2/CXCR1 with orally active small-molecule inhibitors is a promising therapeutic approach for inhibiting melanoma growth and angiogenesis.

Transforming growth factor-beta signaling at the tumor-bone interface promotes mammary tumor growth and osteoclast activation.

Understanding the cellular and molecular changes in the bone microenvironment is important for developing novel therapeutics to control breast cancer bone metastasis. Although the underlying mechanism(s) of bone metastasis has been the focus of intense investigation, relatively little is known about complex molecular interactions between malignant cells and bone stroma. Using a murine syngeneic model that mimics osteolytic changes associated with human breast cancer, we examined the role of tumor-bone interaction in tumor-induced osteolysis and malignant growth in the bone microenvironment. We identified transforming growth factor-beta receptor 1 (TGF-betaRI) as a commonly upregulated gene at the tumor-bone (TB) interface. Moreover, TGF-betaRI expression and activation, analyzed by nuclear localization of phospho-Smad2, was higher in tumor cells and osteoclasts at the TB interface as compared to the tumor-alone area. Furthermore, attenuation of TGF-beta activity by neutralizing antibody to TGF-beta or TGF-betaRI kinase inhibitor reduced mammary tumor-induced osteolysis, TGF-betaRI expression and its activation. In addition, we demonstrate a potential role of TGF-beta as an important modifier of receptor activator of NF-kappaB ligand (RANKL)-dependent osteoclast activation and osteolysis. Together, these studies demonstrate that inhibition of TGF-betaRI signaling at the TB interface will be a therapeutic target in the treatment of breast cancer-induced osteolysis.

Cathepsin G enhances mammary tumor-induced osteolysis by generating soluble receptor activator of nuclear factor-kappaB ligand.

Breast cancer commonly causes osteolytic metastases in bone, a process that is dependent on tumor-stromal interaction. Proteases play an important role in modulating tumor-stromal interactions in a manner that favors tumor establishment and progression. Whereas several studies have examined the role of proteases in modulating the bone microenvironment, little is currently known about their role in tumor-bone interaction during osteolytic metastasis. In cancer-induced osteolytic lesions, cleavage of receptor activator of nuclear factor-kappaB ligand (RANKL) to a soluble version (sRANKL) is critical for widespread osteoclast activation. Using a mouse model that mimics osteolytic changes associated with breast cancer-induced bone metastases, we identified cathepsin G, cathepsin K, matrix metalloproteinase (MMP)-9, and MMP13 to be proteases that are up-regulated at the tumor-bone interface using comparative cDNA microarray analysis and quantitative reverse transcription-PCR. Moreover, we showed that cathepsin G is capable of shedding the extracellular domain of RANKL, generating active sRANKL that is capable of inducing differentiation and activation of osteoclast precursors. The major source of cathepsin G at the tumor-bone interface seems to be osteoclasts that up-regulate production of cathepsin G via interaction with tumor cells. Furthermore, we showed that in vitro osteoclastogenesis is reduced by inhibition of cathepsin G in a coculture model and that in vivo inhibition of cathepsin G reduces mammary tumor-induced osteolysis. Together, our data indicate that cathepsin G activity at the tumor-bone interface plays an important role in mammary tumor-induced osteolysis and suggest that cathepsin G is a potentially novel therapeutic target in the treatment of breast cancer bone metastasis.