Kalyan C. Nannuru

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-β-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.

CXCR1 and CXCR2 enhances human melanoma tumourigenesis, growth and invasion

The aggressiveness of malignant melanoma is associated with differential expression of CXCL-8 and its receptors, CXCR1 and CXCR2. However, the precise functional role of these receptors in melanoma progression remains unclear. In this study, we investigate the precise functional role of CXCR1 and CXCR2 in melanoma progression. CXCR1 or CXCR2 were stably overexpressed in human melanoma cell lines, SBC-2 (non-tumourigenic) and A375P (low-tumourigenic) exhibiting low endogenous expression of receptors. Functional assays were performed to study the resulting changes in cell proliferation, motility and invasion, and in vivo tumour growth using a mouse xenograft model. Our data demonstrated that CXCR1- or CXCR2-overexpressing SBC-2 and A375P melanoma cells had enhanced proliferation, chemotaxis and invasiveness in vitro. Interestingly, CXCR1 or CXCR2 overexpression in SBC-2 cells induced tumourigenicity, and A375P cells significantly enhanced tumour growth as examined in vivo. Immunohistochemical analyses showed significantly increased tumour cell proliferation and microvessel density and reduced apoptosis in tumours generated from CXCR1- or CXCR2-overexpressing melanoma cells. CXCR1- or CXCR2-induced modulation of melanoma cell proliferation and migration was observed to be mediated through the activation of ERK1/2 phosphorylation. Together, these studies demonstrate that CXCR1 and CXCR2 play essential role in growth, survival, motility and invasion of human melanoma.

Cathepsin G Enhances Mammary Tumor–Induced Osteolysis by Generating Soluble Receptor Activator of Nuclear Factor-κB 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.

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.

Matrix Metalloproteinase (MMP)-13 Regulates Mammary Tumor–Induced Osteolysis by Activating MMP9 and Transforming Growth Factor-β 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.

Transforming growth factor-β 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‐β receptor 1 (TGF‐βRI) as a commonly upregulated gene at the tumor‐bone (TB) interface. Moreover, TGF‐βRI 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‐β activity by neutralizing antibody to TGF‐β or TGF‐βRI kinase inhibitor reduced mammary tumor‐induced osteolysis, TGF‐βRI expression and its activation. In addition, we demonstrate a potential role of TGF‐β as an important modifier of receptor activator of NF‐κB ligand (RANKL)‐dependent osteoclast activation and osteolysis. Together, these studies demonstrate that inhibition of TGF‐βRI signaling at the TB interface will be a therapeutic target in the treatment of breast cancer‐induced osteolysis. (Cancer Sci 2009; 100: 71–81)

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.

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.

Cathepsin G-mediated enhanced TGF-β 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-β Signaling and Bone Destruction

Increased transforming growth factor-β (TGF-β) signaling has been observed at the tumor-bone interface of mammary tumor–induced osteolytic lesions despite no observed transcriptional up-regulation of TGF-β. To this point, the mechanism for enhanced TGF-β signaling remains unclear. The bulk of TGF-β that is released at the tumor-bone interface is in an inactive form secondary to association with β-latency–associated protein and latency TGF-β binding protein. We hypothesized that the observed increase in TGF-β signaling is due to increased cathepsin G–dependent, matrix metalloproteinase 9 (MMP9)–mediated activation of latent TGF-β. MMP9 is capable of activating latent TGF-β, and we observed that decreased production of MMP9 was associated with reduced TGF-β signaling. Similar to TGF-β, 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-β to active TGF-β, and reduced the level of TGF-β 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-β, and active TGF-β 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-β signaling at the tumor-bone interface of mammary tumor–induced osteolytic lesions and have identified these proteases as potential therapeutic targets. (Mol Cancer Res 2009;7(8):1224–33)