CTSL inhibitors(Roche)

EpCAM (Epithelial cell adhesion molecule) is often dysregulated in epithelial cancers. Prior studies implicate EpCAM in the regulation of oncogenic signaling pathways and epithelial-to-mesenchymal transition. It was recently demonstrated that EpCAM contains a thyroglobulin type-1 (TY-1) domain. Multiple proteins with TY-1 domains are known to inhibit cathepsin-L (CTSL), a cysteine protease that promotes tumor cell invasion and metastasis. Analysis of human cancer sequencing studies reveals that somatic EpCAM mutations are present in up to 5.1% of tested tumors.

The Catalogue of Somatic Mutations in Cancer (COSMIC) database was queried to tabulate the position and amino acid changes of cancer associated EpCAM mutations. To determine how EpCAM mutations affect cancer biology we studied C66Y, a damaging TY-1 domain mutation identified in liver cancer, as well as 13 other cancer-associated EpCAM mutations. In vitro and in vivo models were used to determine the effect of wild type (WT) and mutant EpCAM on CTSL activity and invasion. Immunoprecipitation and localization studies tested EpCAM and CTSL protein binding and determined compartmental expression patterns of EpCAM mutants.

We demonstrate that WT EpCAM, but not C66Y EpCAM, inhibits CTSL activity in vitro, and the TY-1 domain of EpCAM is responsible for this inhibition. WT EpCAM, but not C66Y EpCAM, inhibits tumor cell invasion in vitro and lung metastases in vivo. In an extended panel of human cancer cell lines, EpCAM expression is inversely correlated with CTSL activity. Previous studies have demonstrated that EpCAM germline mutations can prevent EpCAM from being expressed at the cell surface. We demonstrate that C66Y and multiple other EpCAM cancer-associated mutations prevent surface expression of EpCAM. Cancer-associated mutations that prevent EpCAM cell surface expression abrogate the ability of EpCAM to inhibit CTSL activity and tumor cell invasion.

These studies reveal a novel role for EpCAM as a CTSL inhibitor, confirm the functional relevance of multiple cancer-associated EpCAM mutations, and suggest a therapeutic vulnerability in cancers harboring EpCAM mutations.

Multidrug resistance (MDR) in non-small cell lung cancer (NSCLC) causes chemotherapy failure, with lung cancer stem cells (LCSCs) playing a central role in the development of MDR. Although cathepsin L (CTSL) is known to be associated with tumor progression, its function in LCSCs and MDR remains unclear. The chemotherapeutic sensitivities of spheroid from NSCLC cell lines were evaluated using the CCK-8 assay. Western blot and qPCR analyses were performed to assess the expression levels of CTSL, stem cell markers (CD133 and CD44), stemness-maintaining molecules (OCT4 and SOX2), drug resistance proteins (MDR1 and ABCG2). In vivo experiments were conducted to validate the chemosensitizing effects of CTSL inhibitor, while the relationship between CTSL and the secretion levels of hepatocyte growth factor activator (HGFAC) and HGF was analyzed by RNA-seq, ELISA, qPCR and ChIP-qPCR methods. Our results demonstrated that spheroid cells exhibited pronounced MDR. High expression of CTSL correlated with poor prognosis in patients with NSCLC and enhanced MDR in NSCLC spheroids. Interfering with CTSL increased the sensitivity of NSCLC spheroids to multiple chemotherapy drugs while reducing cell stemness and survival. Moreover, the combination of CTSL inhibitor and docetaxel effectively suppressed tumor growth in vivo. Additionally, there is a positive correlation between CTSL and HGFAC at the mRNA level, and CTSL has a direct transcriptional regulatory effect on HGFAC. Finally, CTSL mediates chemotherapy resistance by regulating the expression of HGFAC/HGF/Met signaling pathway. Taken together, our findings establish CTSL as a pivotal driver of MDR in NSCLC, highlighting its promise as a therapeutic target to overcome this challenge.