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

Abstract

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.

Keywords: KGP94; bone resorption; cathepsin L; metastasis; prostate cancer.

Figure 1

CTSL ablation reduces metastatic disease burden. (a) CTSL semiquantitiative RT-PCR using Tissuescan prostate tissue panels. CTSL mRNA levels were normalized to β-actin. Line, median; whiskers, values at 25th and 75th percentiles; Mann-Whitney test. (b) CTSL secretion by metastatic prostate cancer cell lines. Bars represent means ± s.e.m from three independent experiments. (c) Invasion assay testing the effect of CTSL deficiency on the invasive capacity of PC-3ML cells. Bars represent means ± s.e.m from three independent experiments; Student’s t test. (d) Representative images of invaded cells from c, scale bar= 2 mm. (e) In vivo bone metastasis assay. Measurement of bone metastasis burden based on bioluminescence intensity (photons per second); n ≥ 10. Results are means ± s.e.m, analysis of variance. (f) Representative bioluminescence images of median mice inoculated with empty vector or CTSL shRNA transfected PC-3ML cells. (g) Kaplan-Meier survival curves of mice inoculated with empty vector of CTSL shRNA transfected PC-3ML cells; log-rank test.

Figure 2

KGP94 treatment decreases metastatic tumor burden and improves overall survival. (a) In vivo bone metastasis assay. Representative bioluminescence images of median mice from control and KGP94 treated cohorts. (b) Bone metastases burden in control and KGP94 treated mice measured based on photon flux (photons per second); n≥8. Results are means ± s.e.m, analysis of variance. (c) Kaplan-Meier survival curves of bone metastases bearing mice treated with or without 20 mg/kg KGP94; log-rank test. (d) Representative GFP and H&E images of bone metastases from each experimental group, scale bar= 1 mm. (e) Number of macroscopic metastases in untreated and KGP94 treated mice; n ≥ 8. Line, median; Mann-Whitney test. (f) Distribution of metastases by site.

Figure 3

CTSL inhibition suppresses prostate tumor angiogenesis. (a) Intradermal assay assessing the effect of pharmacological or genetic suppression of CTSL on PC-3ML tumor cell induced angiogenesis; n ≥ 11. EV, empty vector; KD-1, CTSL knockdown PC-3ML tumor nodules; line, median; Mann-Whitney test. (b) Representative images of tumor nodules and blood vessels from a. (c) **, p < 0.005; ***, p< 0.001; Student’s t test. (d) Effect of KGP94 on purified CTSL or conditioned media (CM) stimulated tube forming capacity of HMVEC-L cells. KD CM, conditioned media from CTSL knockdown PC-3ML cells. ***, p< 0.0001, Student’s t test. (e) Representative images from each experimental group shown in d.

Figure 4

KGP94 suppresses bone resorptive capacity of osteoclasts. (a) Quantification of TRAP+ multinucleate osteoclasts 4 days after stimulation with 35 ng/mL RANKL in the presence or absence of KGP94. Bars represent means ± s.e.m from three independent experiments; Student’s t test. (b) Representative images from each experimental group. (c) Western blot analysis of the effect of KGP94 on RANKL stimulated NFκB pathway in RAW 264.7 cells. (d and e) Relative expression of osteoclastogenesis marker genes in RANKL stimulated RAW264.7 cells in the presence or absence of KGP94. Bars represent means ± s.e.m from three independent experiments; analysis of variance. (f) Effect of KGP94 on osteoclast precursor cell viability; Student’s t test. (g) Representative images of bone slices stained with O-toluidine to evaluate the extent of pit formation by osteoclasts under indicated conditions. (h) Percent area of bone resorbed by RANKL stimulated osteoclasts treated with or without KGP94. Results are means ± s.e.m from three independent experiments; Student’s t test.

Figure 5

CTSL promotes osteoclastogenesis. (a) Quantification of CTSL secretion by RAW 264.7 cells during the osteoclast formation process. (b) Dose response analysis of various concentrations of RANKL on the expression of osteoclastogenesis marker genes. (c and d) Effect of CTSL on RANKL stimulated osteoclastogenesis marker expression. Bars represent means ± s.e.m from three independent experiments, Student’s t test. (e) Effect of purified CTSL on osteoclast precursor cell proliferation; Student’s t test. (f) Quantification of TRAP + multinucleate osteoclasts 4 days after stimulation with indicated concentrations of purified CTSL and RANKL. Bars represent means ± s.e.m from three independent experiments, Student’s t test. (g) Representative images from each experimental group.

Figure 6

Impact of CTSL on prostate cancer cell dissemination. Tumor secreted CTSL promotes metastatic progression by activating invasive cascades, stimulating tumor angiogenesis and fostering interaction between metastatic cells and the bone microenvironment. KGP94 mediated CTSL inactivation interferes with these processes and thus impairs metastatic disease progression and associated skeletal morbidities.