Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth

Bone metastasis is common during breast cancer progression. Matrix metalloproteinase-2 (MMP-2) is significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor or host derived MMP-2 contributes to breast cancer growth and does so by processing substrates including type I collagen and transforming growth factorβ (TGFβ) latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. In vitro, BMMPIs impacted the viability of breast cancer cell lines and osteoclast precursors but not osteoblasts. In vivo, we demonstrated using two bone metastatic models (PyMT-R221A and 4T1) that BMMPI treatment significantly reduced tumor growth and tumor associated bone destruction. Additionally, BMMPIs are superior in promoting tumor apoptosis compared to the standard of care bisphosphonate, zoledronate. We demonstrated MMP-2 selective inhibition in the bone microenvironment using specific and broad spectrum MMP probes. Further, compared to zoledronate, BMMPI treated mice had significantly lower levels of TGFβ signaling and MMP generated type I collagen carboxy-terminal (ICTP) fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. We posit that BMMPIs could be easily translated to the clinical setting for the treatment of bone metastases given the well-tolerated nature of bisphosphonates

In vitro comparison of new bisphosphonic acids and zoledronate effects on human gingival fibroblasts viability, inflammation and matrix turnover

Bisphosphonates (BPs) are well known clinically used drugs, commonly applied to treat osteoclast-mediated bone resorption. Some clinically used BPs were demonstrated to be able to inhibit the activity of matrix metalloproteinases (MMPs) (1), a protease family required to fully degrade all the components of the extracellular matrix during connective tissue remodelling (2). Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present work is to compare the effects on cell adhesion, cytotoxicity, inflammatory response occurrence and matrix turnover process in an in vitro model of primary human gingival fibroblasts (HGFs) treated with newly synthesized sulfonamide BPs and with zoledronic acid (ZA), a clinically used drug. Western blot was used to measure Procollagen I, β1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability, LDH was performed for toxicity evaluation, ELISA for Prostaglandin E2 (PGE2) secretion assessment. When compared with ZA, the treatment with the newly synthetized compounds shows increasing viability, Procollagen I expression and decreased expression of β1 integrin in HGFs. Higher levels of released LDH, PGE2 and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthetized compounds-treated samples.These findings imply that new BPs could accelerate the physiological matrix turnover, they are more able to preserve the soft tissue surrounding bone as they have neither inflammatory effects nor toxicity, along with reduced effects on the cell viability, which are instead typical side effects of ZA administration. We can conclude that the newly synthesized compounds are better tolerated, leading to the hypothesis that their use leads to connective tissues side effects reduction compared to clinically used drugs, even though several studies are required to deeply investigate the signaling cascades involved in the mechanism of action of these new BPs

Cutting to the Chase: How Matrix Metalloproteinase-2 Activity Controls Breast-Cancer-to-Bone Metastasis

Bone metastatic breast cancer is currently incurable and will be evident in more than 70% of patients that succumb to the disease. Understanding the factors that contribute to the progression and metastasis of breast cancer can reveal therapeutic opportunities. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes whose role in cancer has been widely documented. They are capable of contributing to every step of the metastatic cascade, but enthusiasm for the use of MMP inhibition as a therapeutic approach has been dampened by the disappointing results of clinical trials conducted more than 20 years ago. Since the trials, our knowledge of MMP biology has expanded greatly. Combined with advances in the selective targeting of individual MMPs and the specific delivery of therapeutics to the tumor microenvironment, we may be on the verge of finally realizing the promise of MMP inhibition as a treatment strategy. Here, as a case in point, we focus specifically on MMP-2 as an example to show how it can contribute to each stage of breast-cancer-to-bone metastasis and also discuss novel approaches for the selective targeting of MMP-2 in the setting of the bone-cancer microenvironment

Cutting to the Chase: How Matrix Metalloproteinase-2 Activity Controls Breast-Cancer-to-Bone Metastasis

Bone metastatic breast cancer is currently incurable and will be evident in more than 70% of patients that succumb to the disease. Understanding the factors that contribute to the progression and metastasis of breast cancer can reveal therapeutic opportunities. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes whose role in cancer has been widely documented. They are capable of contributing to every step of the metastatic cascade, but enthusiasm for the use of MMP inhibition as a therapeutic approach has been dampened by the disappointing results of clinical trials conducted more than 20 years ago. Since the trials, our knowledge of MMP biology has expanded greatly. Combined with advances in the selective targeting of individual MMPs and the specific delivery of therapeutics to the tumor microenvironment, we may be on the verge of finally realizing the promise of MMP inhibition as a treatment strategy. Here, as a case in point, we focus specifically on MMP-2 as an example to show how it can contribute to each stage of breast-cancer-to-bone metastasis and also discuss novel approaches for the selective targeting of MMP-2 in the setting of the bone-cancer microenvironment

Phosphonate Emerging Zinc Binding Group in Matrix Metalloproteinase Inhibitors

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases, capable to degrade the extracellular matrix (ECM) in physiologic conditions. Because of their overexpression and pivotal role in many pathological events, they have been proposed as a therapeutic and prognostic target for a number of diseases. Selectivity among MMPs is essential for realizing the clinical potential of inhibitors. The design of MMP inhibitors (MMPIs) has largely focused on development of various compounds containing a zinc binding group (ZBG) in their structure, with hydroxamate being the most potent one. Due to the high degree of homology in the catalytic domain of all the MMPs, the specificity and selectivity of first generation hydroxamate MMPIs were minimal, with several off-target effects and binding to other metzincins. This review highlights the role of phosphonate as ZBG in the design and development of new MMPIs

Abstract 398: Specific skeletal targeting of MMP-2 inhibitors for the treatment of bone metastatic breast cancer

Background. Bone metastatic breast cancer promotes extensive bone destruction/osteolysis and is currently incurable. Progression of the disease is critically dependent on cancer-bone interaction. Defining the molecular mechanisms underlying this communication can lead to the identification of new therapeutic targets that will eradicate the disease. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed that matrix metalloproteinases (MMPs) such as MMP-2 are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 highlighted the importance of this MMP in driving the growth of the osteolytic breast cancer lesions. We subsequently found that MMP-2 regulation of transforming growth factor β (TGF β) bioavailability was a major mechanism through which MMP-2 mediated this effect. These data support the rationale for the development of selective MMP inhibitors and imply that MMP-2 inhibition would be a successful strategy for the eradication of active bone metastatic breast cancer. Methods. To address systemic dose limiting side effects noted in previous broad spectrum MMP inhibitor trials, we utilized a novel chemical approach to generate bone-targeting, highly selective MMP-2 inhibitors grafted onto a bisphosphonic backbone. In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were inoculated with either luciferase expressing 4T1 or PyMT (100,000) cells. Mice (n = 10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via an activatable MMP probe. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p<0.05) compared to control. In vivo BMMPIs significantly reduced the growth of bone metastatic breast cancer compared to control and the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p<0.05). Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer

Selective inhibition of matrix metalloproteinase-2 in the multiple myeloma-bone microenvironment

Multiple myeloma is a plasma cell malignancy that homes aberrantly to bone causing extensive skeletal destruction. Despite the development of novel therapeutic agents that have significantly improved overall survival, multiple myeloma remains an incurable disease. Matrix metalloproteinase-2 (MMP-2) is associated with cancer and is significantly overexpressed in the bone marrow of myeloma patients. These data provide rationale for selectively inhibiting MMP-2 activity as a multiple myeloma treatment strategy. Given that MMP-2 is systemically expressed, we used novel “boneseeking” bisphosphonate based MMP-2 specific inhibitors (BMMPIs) to target the skeletal tissue thereby circumventing potential off-target effects of MMP-2 inhibition outside the bone marrow-tumor microenvironment. Using in vivo models of multiple myeloma (5TGM1, U266), we examined the impact of MMP-2 inhibition on disease progression using BMMPIs. Our data demonstrate that BMMPIs can decrease multiple myeloma burden and protect against cancer-induced osteolysis. Additionally, we have shown that MMP-2 can be specifically inhibited in the multiple myeloma-bone microenvironment, underscoring the feasibility of developing targeted and tissue selective MMP inhibitors. Given the well-tolerated nature of bisphosphonates in humans, we anticipate that BMMPIs could be rapidly translated to the clinical setting for the treatment of multiple myeloma