Dhivya R. Sudhan

Cathepsin L targeting in cancer treatment.

Proteolytic enzymes may serve as promising targets for novel therapeutic treatment strategies seeking to impede cancer progression and metastasis. One such enzyme is cathepsin L (CTSL), a lysosomal cysteine protease. CTSL upregulation, a common occurrence in a variety of human cancers, has been widely correlated with metastatic aggressiveness and poor patient prognosis. In addition, CTSL has been implicated to contribute to cancer-associated osteolysis, a debilitating morbidity affecting both life expectancy and the quality of life. In this review, we highlight the mechanisms by which CTSL contributes to tumor progression and dissemination and discuss the therapeutic utility of CTSL intervention strategies aimed at impeding metastatic progression and bone resorption.

Cathepsin L inhibition by the small molecule KGP94 suppresses tumor microenvironment enhanced metastasis associated cell functions of prostate and breast cancer cells

Metastasis remains the major cause of therapeutic failure, poor prognosis and high mortality in breast and prostate cancer patients. Aberrant microenvironments including hypoxia and acidic pH are common features of most solid tumors that have been long associated with enhanced metastasis and poor patient outcomes. Novel approaches to reduce metastatic incidences and improve overall survival of cancer patients clearly are needed. The crucial role of Cathepsin L (CTSL) in the dissemination of tumor cells has led to the development of novel cathepsin L inhibition strategies. The present study evaluated the ability of KGP94, a small molecule inhibitor of CTSL, to impair the metastatic phenotype of prostate (PC-3ML) and breast (MDA-MB-231) cancer cells both under normal and aberrant microenvironmental conditions. To assess the role of CTSL in hypoxia and acidosis triggered metastasis associated cell functions, secreted CTSL levels were determined under conditions pertinent to the tumor microenvironment. Acute exposures to hypoxic or acidic conditions significantly elevated secreted CTSL levels either through an increase in intracellular CTSL levels or through activation of lysosomal exocytosis or both, depending on the tumor type. Increases in CTSL secretion closely paralleled enhanced tumor cell migration and invasion suggesting that CTSL could be an essential factor in tumor microenvironment triggered metastasis. Importantly, KGP94 treatment led to marked attenuation of tumor cell invasion and migration under both normal and aberrant microenvironmental conditions suggesting that it may have significant utility as an anti-metastatic agent.

Cathepsin L inactivation leads to multimodal inhibition of prostate cancer cell dissemination in a preclinical bone metastasis model

It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra‐cardiac injection of human PC‐3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients.

Therapeutic potential of adipose stem cell-derived conditioned medium against pulmonary hypertension and lung fibrosis.

Pulmonary hypertension (PH) and pulmonary fibrosis (PF) are life threatening cardiopulmonary diseases. Existing pharmacological interventions have failed to improve clinical outcomes or reduce disease‐associated mortality. Emerging evidence suggests that stem cells offer an effective treatment approach against various pathological conditions. It has been proposed that their beneficial actions may be mediated via secretion of paracrine factors. Herein, we evaluated the therapeutic potential of conditioned media (CM) from adipose stem cells (ASCs) against experimental models of PH and PF.

Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L.

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates.

Abstract 4030: In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model

It is estimated that ∼85% of patients with advanced prostate cancer develop skeletal metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of the prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Tumor cells rely on proteolytic enzymes for successful establishment of metastases. Cathepsin L (CTSL) is a lysosomal cysteine protease that is up regulated in a wide range of human cancers including prostate cancer. The elevated secretion of CTSL by aggressively metastasizing tumor cells plays a key role in metastatic spread of tumor cells through proteolytic degradation of extracellular matrix and basement membrane components. In addition, tumor secreted cytokines have been shown to increase osteoclastic secretion of CTSL to promote bone resorption. Intervention strategies targeting CTSL may therefore serve a dual purpose by inhibiting both metastatic dissemination as well as bone resorption. The goal of present studies was to evaluate the anti-metastatic and anti-bone resorptive efficacy of CTSL inhibition using the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94). KGP94 treatment significantly impaired the metastatic phenotype of highly metastatic prostate cancer PC-3ML cells. Treatment with 25µM KGP94 decreased the migratory and invasive capacities of PC-3ML cells by 78 and 53% respectively. To validate the anti-metastatic efficacy of KGP94 in-vivo, luciferase labelled PC-3ML cells were injected into the left ventricle of male nu/nu mice followed by daily treatment with 20mg/kg KGP94. Metastatic progression was monitored by weekly bioluminescence imaging using firefly luciferase reporter system. KGP94 treatment led to a significant reduction in metastatic tumor burden (65% reduction) and a reduction in metastatic incidence. Histological assessment of bones further confirmed the reduction in metastatic burden upon KGP94 treatment. CTSL inhibition also led to a significant increase in overall survival of tumor bearing mice (p=0.01). In vitro assessment of anti-resorptive function of KGP94 was performed using osteoclastogenesis and pit formation assays. KGP94 treatment of RAW264.7 osteoclast precursor cells led to a significant decrease (72%) in osteoclast formation as demonstrated by a decrease in the number of TRAP+ multinucleate cells. KGP94 also inhibited the bone resorptive activity of osteoclasts as indicated by a significant reduction in the number of resorptive pits formed by osteoclasts on bone slices. In conclusion, our findings suggest that CTSL inhibition using small molecule KGP94 can significantly impair metastatic dissemination of prostate cancer cells and alleviate bone resorption. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4030. doi:10.1158/1538-7445.AM2014-4030

Abstract B150: Cathepsin L targeting abrogates tumor microenvironment enhanced metastatic potential in prostate cancer cells.

Metastatic dissemination of tumor cells remains the primary cause of therapeutic failure and high mortality in men with prostate cancer. The development of novel therapeutic strategies that impair the metastatic process therefore is critical to improving prostate cancer patient survival. Tumor cells rely on proteolytic enzymes for successful establishment of metastases. Cathepsin L (CTSL) is a lysosomal cysteine protease that is up regulated in a wide range of human cancers including prostate cancer. The elevated secretion of CTSL by aggressively metastasizing tumor cells plays a key role in metastatic spread of tumor cells through proteolytic degradation of extracellular matrix and basement membrane components. Thus intervention strategies targeting CTSL may provide a novel anti-metastatic therapeutic approach to improve treatment outcome. This concept is supported by recent studies in our laboratory which showed that treatment with the specific small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) significantly impaired the migratory and invasive potential of metastatic prostate cancer cells. Clinical and experimental observations emphasize that pathophysiological tumor microenvironments characterized by hypoxia and acidic pH augment metastatic aggressiveness. Thus, the goal of the present studies was to explore the role of CTSL in tumor microenvironment-triggered elevation of invasiveness and to elucidate whether the administration of KGP94 could inhibit the metastatic phenotype in prostate cancer cells. Exposure to 1% oxygen and pH values of 6.8 and 6.4, enhanced the invasive phenotype of both the human prostate cancer cell line PC-3 and its highly metastatic subline PC-3-ML cells 1.5 to 2 fold. Treatment with 10 and 25μM KGP94 suppressed the heightened invasive potential (33% inhibition at 10μM and 60% inhibition at 25μM in PC-3 ML cells). Interestingly upon KGP94 treatment, invasiveness of these cells reduced below basal invasion levels observed under normoxic conditions. Aberrant microenvironment induced enhancement of PC-3 and PC-3-ML invasive potential was attributable to elevated activity of secreted CTSL. Molecular assessment revealed that the observed increase in CTSL activity was a consequence of exocytosis of lysosomes containing active CTSL into the extracellular milieu. Lysosomal/endosomal marker LAMP-1immunostaining revealed that in contrast to the peri-nuclear localization observed in control cells, lysosomes in cells exposed to low oxygen tension and pH traffic towards the cell periphery. Observations based on immunostaining were confirmed through biochemical quantification of the release of lysosomal enzyme β-Hexosaminidase into the media after hypoxic or acidic exposures. In conclusion, our findings suggest that hypoxia and acidosis triggered elevation of PC-3 and PC-3-ML invasive potential is associated with CTSL activation. KGP94 mediated CTSL inhibition thus effectively impaired the pathophysiological tumor microenvironment induced enhancement of the metastatic phenotype of prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B150.

Abstract 4185: Role of Cathepsin L in breast cancer angiogenesis

Recognition of the dependence of angiogenesis on proteolytic enzymes has raised significant interest in evaluation of proteases as potential targets to disrupt tumor angiogenesis. While the role of cysteine proteases in tumor metastasis is well understood, their contribution to tumor angiogenesis remains less explored. Cysteine cathepsins, in particular cathepsin L (CTSL) gets are upregulated in a wide range of human cancers. In transformed cells extracellular CTSL levels can increase up to 200-fold and comprise up to 40% of total secreted proteins. The role of CTSL in promoting tumor metastasis is supported by recent studies in our laboratory which have shown that treatment with the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) significantly impairs the metastatic phenotype of prostate and breast cancer cells. However, the contribution of CTSL to tumor angiogenesis remains less well explored. Thus, the goal of the present studies was to evaluate the role of tumor cell secreted CTSL in tumor cell induced angiogenesis. Upon activation by pro-angiogenic stimuli, endothelial cells migrate and invade through the interstitium to form tubes. The effect of CTSL on the migratory, invasive and tube formation capacity of endothelial cells was therefore investigated. Stimulation of human micro-vascular endothelial cells of the lung (HMVEC-L) with purified human CTSL led to a dose dependent increase in endothelial cell invasion (1.5-fold at 10ng/ml and 2-fold at 100ng/ml). Furthermore, CTSL (100ng/ml) increased the tube forming ability of endothelial cells as demonstrated by an increase in total tube length. Similarly, incubation of HMVEC-L with conditioned media from MDA-MB-231 breast cancer cells, led to a significant increase in their migration, invasion and tube formation capacity (migration 1.3-fold; invasion 2.5-fold). Compared to parental MDA-MB-231 breast cancer cells, CTSL knockdown cells that were inoculated intradermally into ventral skin flaps of female nude mice showed a significant reduction in their ability to induce blood vessel formation (3.5-fold).These proangiogenic endothelial cell functions were accompanied by a significant increase in endothelial cell proliferation upon stimulation with purified CTSL (2-fold). Microarray analyses revealed a significant upregulation of cell cycle related genes by CTSL. Western blot analyses further confirmed upregulation of members of the cyclin family by CTSL. In conclusion, our findings suggest that in addition to its pro-metastatic function, CTSL might further contribute to tumor progression by eliciting various pro-angiogenic endothelial cell functions. Citation Format: Dhivya Sudhan, Belen Rabaglino, Charles Wood, Dietmar Siemann. Role of Cathepsin L in breast cancer angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2015-4185

Abstract A33: Small molecule cathepsin L inhibitor KGP94 suppresses hypoxia and acidosis potentiated metastatic capacity of prostate and breast cancer cells

Tumor metastasis is the primary cause of treatment failure and high mortality rates in prostate and breast cancer patients. The development of novel therapeutic strategies that impair the metastatic process therefore is critical to improving prostate and breast cancer patient survival. Elevated cathepsin L (CTSL) secretion by tumor cells has been correlated with metastatic aggressiveness and with poor patient prognosis. Metastatic occurrence can be exacerbated by hypoxic and acidic tumor microenvironments. The goals of the present studies were to explore the role of CTSL in tumor microenvironment-triggered elevation of invasiveness and to elucidate whether the administration of the CTSL specific small molecule inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) could inhibit the enhanced metastatic phenotype in human prostate and breast cancer cells. Comparison of CTSL secretion levels in a number of prostate and breast cancer cells of varying invasive capacities revealed a positive correlation between CTSL secretion and invasive potential. Highly metastatic prostate and breast cancer cell lines PC-3ML and MDA-MB-231 respectively, were used for further studies. In response to treatment with 10μM and 25μM of KGP94 under normoxic conditions, PC-3ML and MDA-MB-231 showed a marked decrease in migratory and invasive potential. These results occurred in the absence of cytotoxicity as determined using a clonogenic cell survival assay. Aberrant microenvironmental conditions (hypoxia; 1%O2 or acidosis; pH, 6.8) enhanced invasiveness of PC-3ML and MDA-MB-231 cells in concert with elevated secretion of CTSL. Molecular assessments revealed that the observed increase in CTSL secretion was a consequence of increased CTSL expression and exocytosis of lysosomes containing active CTSL into the extracellular milieu. Immunostaining with the lysosomal/endosomal marker LAMP-1 revealed that in contrast to the peri-nuclear localization observed in control cells, lysosomes in cells exposed to low oxygen tension or pH traffic toward the cell periphery. Observations based on immunostaining were confirmed through biochemical quantification of the release of lysosomal enzyme β-Hexosaminidase into the incubation media after hypoxic or acidic exposures. KGP94 treatment of PC-3ML and MDA-MB-231 cells exposed to aberrant microenvironmental conditions reduced the enhanced invasive phenotype of these cells below basal invasion levels observed under normoxic conditions. Our findings indicate that (i) the hypoxia and acidosis potentiated metastatic phenotype of PC-3ML and MDA-MB-231 cells is associated with elevated CTSL secretion and (ii) KGP94 mediated CTSL inhibition effectively impairs the microenvironmentally induced enhancement of the metastatic phenotype of prostate and breast cancer cells. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. Small molecule cathepsin L inhibitor KGP94 suppresses hypoxia and acidosis potentiated metastatic capacity of prostate and breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A33.

Abstract 5093: Tumor angiogenesis: The role of Cathepsin L and its therapeutic intervention by the small molecule inhibitor KGP94.

Angiogenesis, the process of new blood vessel formation from pre-existing ones, is crucial to tumor growth and progression. Understanding the interplay between tumor cells and surrounding vasculature is of significant interest towards the identification of novel pro-angiogenic factors and the development of anti-angiogenic therapies. Cathepsin L (CTSL) is a cysteine protease that is up regulated in a wide range of human cancers. In transformed cells extracellular CTSL levels can increase up to 200-fold and comprise up to 40% of total secreted proteins. The role of CTSL in promoting tumor metastasis is supported by recent studies in our laboratory which have shown that treatment with the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) significantly impairs the metastatic phenotype of prostate and breast cancer cells. However, the contribution of CTSL to tumor angiogenesis remains less well explored. Thus, the goal of the present studies was to evaluate the role of tumor cell secreted CTSL in tumor cell induced angiogenesis and to determine whether KGP94 treatment could inhibit this process. Upon activation by pro-angiogenic stimuli, endothelial cells migrate and invade through the interstitium to form tubes. The effect of CTSL on the migratory, invasive and tube formation capacity of endothelial cells was therefore investigated. Stimulation of human micro-vascular endothelial cells of the lung (HMVEC-L) with purified human CTSL led to a dose dependent increase in endothelial cell invasion (1.5 fold at 10ng/ml and 2 fold at 100ng/ml). Furthermore, CTSL (100ng/ml) increased the tube forming ability of endothelial cells as demonstrated by an increase in total tube length, average length of individual tube and number of tubes formed. Tumor cells secrete high levels of CTSL; therefore the effect of tumor cell conditioned media on endothelial cell migratory and invasive potential was evaluated. Incubation of HMVEC-L with conditioned media from MDA-MB-231 breast cancer cells, led to a significant increase in their migration, invasion and tube formation capacity (migration 1.3 fold; invasion 2.5 fold). These pro-angiogenic endothelial cell functions were significantly impaired in HMVEC-L treated with 10 or 25μM KGP94. For example, there was a 55% reduction in conditioned media stimulated invasiveness at 10μM and a 68% reduction at 25μM. Similarly, KGP94 treatment also significantly impaired purified CTSL and conditioned media stimulated tube formation. Interestingly, KGP94 treatment alone, did not affect the migratory or invasive capacity of unstimulated endothelial cells, suggesting that this agent might selectively impede tumor induced angiogenesis. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. Tumor angiogenesis: The role of Cathepsin L and its therapeutic intervention by the small molecule inhibitor KGP94. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2013-5093