Type Ⅱ Transmembrane Serine Proteases In Hematological Malignancies

Objective: The type Ⅱ transmembrane serine protease (TTSP) family includes a groupof trypsin-like enzymes that are anchored on the cell surface via an integraltransmembrane domain near the N-terminus. Many TTSPs have been found to play animportant role in a variety of physiological and pathological processes. Dysregulation ofthese TTSPs may lead to serious diseases such as cancer, anemia, hearing loss, andcardiovascular disease. There are17TTSP members in humans. Matriptase is one of theTTSPs and plays a vital role in normal epithelia physiology. Under pathologicalconditions, matriptase deficiency or overexpression may contribute to skin disordersand solid tumors. To date, however, little is known about the biological function ofTTSPs in hematological malignancies. The goal of this study is to examine theexpression of TTSPs in hematological malignancies. Based on our preliminary results,we selected matriptase as a candidate and studied its role in chronic lymphocyticleukemia (CLL).Methods:(1) Reverse transcription PCR (RT-PCR) was used to analyze mRNA expression of17TTSPs in15human hematological cancer cell lines and81bone marrow samplesfrom patients with hematological malignancies.(2) Quantitative real-time PCR (qRT-PCR) was used to quantify matriptase and itsinhibitor, hepatocyte growth factor activator inhibitor-1(HAI-1), mRNA expression inbone marrow cells from patients with different types of leukemia.(3) Western blotting was performed to examine matriptase protein expression inhuman hematological cancer cell lines and bone marrow cells from leukemia patients.(4) Flow cytometry and immunostaining were used to determine the cell membraneexpression of matriptase in B cell-derived Namalwa cells and bone marrow cells from leukemia patients.(5) Small hairpin RNA (shRNA) interference and recombinant matriptase inhibitor,HAI-1, were used to inhibit matriptase expression and activity in Namalwa and CLLcells to test the effect of matriptase inhibition on cancer cell invasion, migration andproliferation in cell-based assays.Results：(1) By RT-PCR, we found that matriptase and human airway trypsin-likeprotease-like4(HATL4) mRNA expression was highly up-regulated in different typesof hematological malignancies. Matriptase mRNA expression was highly up-regulatedin B cell-derived lymphoma cell lines (Namalwa and Raji) and bone marrow cells frompatients with CLL. HATL4mRNA was overexpressed in bone marrow cells frompatients with acute myeloid leukemia (AML). We decided to focus on matritpase tofurther study its role in CLL.(2) By qRT-PCR, we showed that matriptase mRNA expression in bone marrow cellsfrom patients with CLL was~22fold higher than that in normal peripheral blood (NPB)and normal bone marrow (NBM) cells. Matriptase mRNA expression was mostlyundetectable in other types of leukemia cells, such as acute lymphocytic leukemia(ALL), acute myelocytic leukemia (AML) and chronic myelocytic leukemia (CML). Inthese CLL cells, the matriptase inhibitor HAI-1expression was barely detectable,indicating that matriptase activity was likely increased in cancer cells in CLL patients.(3) By Western blotting, we detected high levels of matriptase protein in Bcell-derived Namalwa and Raji cells and bone marrow cells from CLL patients. Theresult was consistent with the findings in PCR-based experiments.(4) By immunostaining, we showed that matriptase protein was present on thesurface of Namalwa cells and CLL cells from patients. The results were confirmed byflow cytometry, which showed that matriptase was positive in~99%of Namalwa cellsand~93%of bone marrow cells from CLL patients. As a control, matriptase was mostlynegative in NPB cells.(5) Using a specific shRNA against the matriptase gene, we were able to reduce matriptase mRNA and protein expression in Namalwa cells by>80%, as indicated byqRT-PCR and Western analyses. In a transwell invasion assay, we showed that silencingmatriptase expression by shRNA or inhibiting matriptase activity by HAI-1markedlyinhibited Namalwa, Raji and CLL cells in matrigel invasion.(6) Under similar experimental conditions, matriptase inhibition by shRNA orHAI-1did not significantly alter cancer cell migration or proliferation. These dataindicate that matriptase is most likely to promote cancer cell invasion via its proteolyticactivity to degrade matrix proteins.Conclusion: Our studies demonstrate, for the first time, that matriptase wasspecifically up-regulated in CLL. The cell membrane expression indicates thatmatriptase may be used as a cell surface marker for the diagnosis and monitoring ofCLL. We show that cancer cell invasion was markedly reduced when matriptaseexpression was silenced or its activity was inhibited by the inhibitor HAI-1. Our datasuggest that specific matriptase inhibitors may be developed as a new therapy to inhibitor prevent CLL cell invasion and metastasis.