Human Airway Trypsin-like Protease 4(HATL4) In Acute Myeloid Leukemia

Chapter One: HATL4 Expression in AML Patients and Disease PrognosisObjective: The type II transmembrane serine protease(TTSP) family includes a group of trypsin-like enzymes that are anchored on the cell surface via the N-terminal transmembrane domain. TTSP members have been found to play an important role in a variety of physiological and pathological processes. Deregulation of these TTSPs may lead to serious diseases such as cancer, anemia, cardiovascular disease and hearing loss. Studies have implicated TTSPs in solid tumors. Previous studies in our laboratory also indicated a role of TTSPs in hematological malignancies. Human airway trypsin-like protease 4(HATL4) is a TTSP family member. Its physiological function remains unclear. Our preliminary studies suggested that HATL4 was abnormally up-regulated in acute myeloid leukemia(AML) cancer cells. The goal of this study is to analyze HATL4 expression in bone marrow cells from AML patients and to examine potential roles of HATL4 in AML cancer cell biology. Our studies may provide new insights into TTSP-mediated mechanisms in the development and progression of hematological malignancies. Our studies may also help to identify novel molecular targets with diagnostic and prognostic values for AML.Methods: 1. Bone marrow cells were obtained from patients with hematological malignancies. White blood cells were isolated for further experiments. 2. Reverse transcription PCR(RT-PCR) was used to analyze HATL4 m RNA expression in human hematological cancer cell lines and bone marrow samples from patients with hematological malignancies. Quantitative real-time PCR(q RT-PCR) was used to quantify HATL4 m RNA expression levels in bone marrow cells from leukemia patients. 3. Western blotting was performed to examine HATL4 protein expression in human hematological cancer cell lines and bone marrow cells from leukemia patients. 4. Flow cytometry and immunostaining were used to examine HATL4 expression on the surface of AML-derived THP-1 cells and bone marrow cells from leukemia patients. 5. Correlation between HATL4 expression and clinical parameters in AML patients was examined.Results: 1. By RT-PCR, HATL4 m RNA was found to be highly expressed in AML-derived(HEL, SHI-1 and THP-1) and chronic myeloid leukemia(CML)-derived(KU-812, MEG-01 and K562) cell lines. HATL4 m RNA overexpression was also found in bone marrow cells from AML patients. The results were confirmed by q RT-PCR using normal peripheral blood(NPB) and normal bone marrow(NBM) cells as controls. In contrast, HATL4 m RNA expression was hardly detectable in bone marrow cells from patients with other types of leukemia, such as CML, acute lymphoblastic leukemia(ALL) and chronic lymphocytic leukemia(CLL). 2. Western blotting detected high levels of HATL4 protein in AML- and CML-derived cells and bone marrow cells from AML patients, which was consistent with the findings from the PCR-based experiments. 3. By immunostaining, we identified HATL4 protein on the surface of THP-1 cells and AML cells from patients. The results were confirmed by flow cytometry-based experiments, in which HATL4 was positive in ~100% of THP-1 cells and ~100% of neutrophils and monocytes in AML patient bone marrows. In contrast, HATL4 was negative in NPB cells. 4. Among 105 bone marrow samples from AML patients after chemotherapy, 19 HATL4 positive samples were from patients with poor- or moderate-risk, and 86 HATL4 negative samples were from patients with better- or moderate-risk. HATL4 expression correlated with minimal residual diseases(MRD) and poor prognostic risks. Consistently, patients who were HATL4 positive had shorter progression-free survival time, as shown by Kaplan-Meier analysis.Conclusions: Our studies show that HATL4 expression is abnormally up-regulated in AML, but not in other types of leukemia cells examined in our study. In AML patients after chemotherapy, HATL4 expression correlated with MRD, poor prognostic risks and shorter progression-free survival time. As HATL4 is a transmembrane protease, our findings suggest that HATL4 may be used as a cell surface marker for the diagnosis and monitoring of AML. HATL4 may also have prognostic values in predicting progression-free survival time in AML patients after chemotherapy.Chapter Two: In Vitro Studies of HATL4 in AML Cancer Cell BiologyObjective: As presented in Chapter One, we found that HATL4 expression was highly up-regulated in bone marrow-derived cancer cells from AML patients and that HATL4 expression was associated with poor clinical outcomes in AML patients, suggesting a role of ectopic HATL4 expression in the pathogenesis of AML. We hypothesized that HATL4 overexpression may alter cancer cell properties, thereby contributing to AML development and progression. In this study, we used AML-derived THP-1 cells that express endogenous HATL4 as a cell model to test if blocking HATL4 expression reduces HTP-1 cell proliferation, migration and invasion in in vitro experiments.Methods: 1. Small hairpin RNA(sh RNA) interference was used to inhibit HATL4 expression in THP-1 cells to test the effect of HATL4 inhibition on cancer cell invasion, migration and proliferation in cell-based assays. 2. Flow cytometry(FCM) was used to detect Green Fluoresent Protein(GFP) marker in sh RNA control and targeted cell groups. Parental THP-1 cells were used as a negative control. 3. A pc DNA3.1 plasmid expressing wild-type(WT) HATL4 was used as a template to construct a HATL4 mutant that was expected to be resistant to the HATL4-targeting sh RNA. The HATL4 mutant plasmid was electroporated into the THP-1 cells, in which the HATL4 gene had been silenced. The resulting cells expressed comparable levels of HATL4 to that in control THP-1 cells in the presence of the HATL4-targeting sh RNA. 4. Cells expressing the mutant HATL4 were sorted by FCM using indirect fluorescent staining. 5. Cell counting kit-8(CCK-8) assay was used to test cell proliferation in THP-1 cells with(targeted cells) or without(control cells) HATL4 gene silencing and in HATL4-silenced THP-1 cells that expressed the sh RNA resistant mutant HATL4(resistant cells). 6. Transwell migration assay was used to test the migration of the three groups of THP-1 cells that were described above. 7. Transwell invasion assay was used to examine the ability of the three groups of THP-1 cells in invading Matrigels. The effects of an MMP inhibitor, GM6001, were also tested in these experiments. 8. Zymography was performed to analyze MMP-2 and MMP-9 activities in the conditioned media from the three groups of THP-1 cells. 9. WT HATL4 was expressed in Chinese Hamster Ovary(CHO) cells by cell transfection. The transfected CHO cells were tested for pro-MMP-2 activation using an MMP-2 activity assay to determine if HATL4 activates pro-MMP-2.Results: 1. Using a specific sh RNA against the HATL4 gene, we were able to reduce HATL4 m RNA and protein expression in THP-1 cells by >70%, as indicated by q RT-PCR and Western analyses. 2. In cell proliferation studies, altered HATL4 expression by gene silencing or expressing an sh RNA resistant mutant did not significantly alter THP-1 cancer cell proliferation. 3. In transwell cell migration studies, inhibition of HATL4 expression did not alter THP-1 cell migration. 4. In transwell Matrigel invasion studies, silencing HATL4 expression by the sh RNA inhibited THP-1 cells in invading Matrigels. The MMP inhibitor GM6001 also inhibited the THP-1 cells of the control and targeted groups. These data indicate that HATL4 may promote cancer cell invasion, possibly, by an MMP-dependent mechanism. 5. Western analysis showed similar levels of pro-MMP-2 in the conditioned media from the three groups of THP-1 cells. However, the levels of activated MMP-2(~62 k Da) and MMP-9(~83 k Da) were significantly reduced in the targeted group. The reduction was particularly greater for activated MMP-2. These results suggested that HATL4 inhibition may reduce MMP-2 zymogen activation in THP-1 cells. 6. In CHO cells, recombinant HATL4 expression enhanced pro-MMP-2 activation, as indicated by a fluorescence-based MMP-2 activity assay, suggesting that HATL4 may directly activate MMP-2.Conclusions: HATL4 expression promoted AML-derived THP-1 cell invasion in Matrigels. The activity appeared to be mediated by an MMP-dependent mechanism. HATL4 may activate MMP-2 and other MMPs in THP-1 cells, thereby promoting extracellular matrix degradation and cancer cell invasion.Chapter Three: Mouse Model Studies of HATL4 in Tumor GrowthObjective: In Chapter One, we found that HATL4 was highly expressed in AML cells and the expression was associated with poor prognosis in AML patients. In Chapter Two, we showed that HATL4 expression promoted AML-derived THP-1 cancer cell invasion. These data indicate a role of HATL4 in AML development and progression. In this Chapter, we verified our findings in a nude mouse model, in which THP-1 cells with altered HATL4 expression were inoculated subcutaneously. We examined the tumor growth in these nude mice. Our goal is determine if HATL4 expression promotes cell proliferation, apoptosis and angiogenesis in THP-1 cell-derived tumors in vivo.Methods: 1. The six groups of THP-1 cells(two each from the control, targeted and resistant groups) were inoculated subcutaneously into male nude mice of six-weeks old. Tumor growth was monitored by measuring tumor volume in live animals. 2. Immunohistochemistry staining was used to examine Ki-67 and CD34 expression in tumor tissues to assess cell proliferation and tumor angiogenesis. 3. Apoptosis was analyzed in tumor tissue sections using the Td T-mediated d UTP nick end labeling(TUNEL) assay.Results: 1. Tumor formation was observed visually in the nude mice on day 10 after inoculation. After 24 days of inoculation, tumor size in the mice injected with HATL4-targeted THP-1 cells was smaller than that in mice with the other two groups of THP-1 cells(control and resistant groups). By day 36, fluorescent signals at the inoculation sites were detected by living animal imaging. The signals were weaker in the mice with the HATL4-targeted THP-1 cells. When the tumors were isolated from the mice, the tumor weight in the mice with the HATL4-targeted THP-1 cells was significantly lower than that in the other two groups of mice. 2. Compared with those in the control and resistant groups, Ki-67 positive cells in tumor tissue sections were less in mice with the HATL4-targeted THP-1 cells. The results suggested that silencing HATL4 expression inhibited THP-1 cell proliferation in tumors in the nude mice and that such a tumor suppression effect probably was mediated by inhibiting THP-1 cell proliferation and/or local cancer cell invasion in vivo. 3. In tumor tissue sections, no significant difference in TUNEL-positive cell number was found among the three groups of nude mice. The results suggested that apoptosis was not significantly altered in the tumors from these mice. Thus, the reduced tumor growth in the mice with the HATL4-targeted THP-1 cells was unlikely caused by enhanced apoptosis in tumor tissues. 4. CD34 staining for vascular marker in tumor tissues was similar in abundance in all three groups of mice, suggesting the HATL4 expression did not significantly alter angiogenesis in the THP-1 cell-derived tumors.Conclusions: HATL4 expression promoted the growth of THP-1 cell-derived tumors in a nude mouse model. The tumor promotion was probably mediated by increased tumor cell proliferation and/or local invasion. In summary, we discovered that HATL4 was highly up-regulated in bone marrow cells in AML patients and that the HATL4 expression correlated MRD and poor prognosis in these patients. In cell-based experiments and nude mouse model studies, we found that HATL4 expression promoted AML-derived cancer cell invasion and tumor growth. Biochemical studies also indicated that HATL4 may activate MMPs to promote extracellular matrix degradation. Our results provide new insights into cell membrane-bound serine proteases in the pathogenesis of AML. Our findings also indicate that HATL4 may represent a novel biomarker with diagnostic and prognostic values for AML.