Tartrate-resistant acid phosphatase/ACP5 as a driver of cancer : dissection of its oncogenic mechanisms and identification of small molecule inhibitors

Abstract

Cancer patients diagnosed with metastasis have an increased risk of dying. To be able to predict and target tumors with an increased risk for spreading, underlying molecular events need to be better dissected and understood. TRAP is a metalloenzyme existing in two isoforms - a precursor form (TRAP 5a) and a highly enzymatically active form (TRAP 5b). TRAP expression was detected in cancer cells of several primary and metastatic tumors and expression levels were raised with increasing malignancy. TRAP expression was further correlated to clinical parameters of aggressiveness such as reduced tumor- and metastasis-free survival. Underlying molecular processes remain unclear and only a limited amount of studies is addressing the respective role of the TRAP isoforms in cancer.

In this thesis, two major milestones were addressed tackling the role of TRAP (isoforms) in cancer cell metastasis. (I) Identification and characterization of previously reported and novel small molecule inhibitors of TRAP (II) Characterization of functional alterations and cellular mechanisms induced by TRAP perturbation.

The TRAP inhibitor 5-phenylnicotinic acid (5-PNA/CD13) was previously identified by fragment-based screening. In Paper I, 5-PNA was further characterized for its ability to inhibit the mammalian TRAP, its selectivity for TRAP and its cytotoxicity in a cellular model. TRAP-dependent migration was inhibited by 5-PNA, shown to be selective for the TRAP 5b isoform. By small molecule screening of a library containing drug-like compounds in Paper II, several inhibitors for TRAP activity were found and selected based on a strict filtration strategy. Orthogonal validation, full-concentration responses and isoform selectivity were assessed for a selection of hit compounds. Six potential lead structures were characterized for molecular docking modes. The compound CBK289001 rendered valid as inhibiting TRAP-dependent migration in a cell system and initial structure-activity relationships were derived. The aggressiveness of cancer cells with perturbations of TRAP expression was assessed by functional studies in Paper III. TRAP had a promotive effect on cancer cell elongation, proliferation, migration and invasion. Proteomics and Phospho-proteomics outlined changes in the cellular network associated with extracellular matrix modulation and cell adhesion, and a regulation of TGFβ and CD44 signaling. A list of potential TRAP substrates was generated. The role of TRAP 5b isoform in cancer cell aggressiveness and Cathepsin K (CtsK) in its generation and processing was investigated in Paper IV. TRAP 5b was significantly increased compared to TRAP 5a in cells overexpressing TRAP. Inhibition of CtsK, an enzyme shown to be able to cleave TRAP, resulted in an intermediate processed TRAP 5b form with similar activity and promotive effect on migration. CtsK colocalized highly with TRAP 5b and cleaving changed the subcellular localization of TRAP 5b.

In summary, the work presented in this thesis is contributing to the knowledge about the role of TRAP in cancer metastasis. Specifically, we were able to show a connection of TRAP 5b to metastasis-related cell functions and the involvement of TGFβ/CD44 signaling. Possible starting points for the development of potent and TRAP-specific inhibitors are provided.