The Role Of PIB5PA Downregulated By Histone Hypoacetylation In Carcinogenesis And Progression Of Human Melanoma And Molecular Mechanisms

Background&Objective Melanoma is the most fatally malignant tumor derived fromskin cells called melanocytes. Since melanoma is highly malignant and easy tometastasize in early stage, most patients have lost the chance of surgery when diagnosedand the effects of chemotherapy and radiotherapy are limited. Therefore, melanomatreatments are focused on the individual therapy for specific molecular targets throughunderstanding the pathogenesis of melanoma and mechanisms of resistance tochemotherapy. It has been reported that PI3K/Akt signaling pathway is constitutivelyactivated in up to70%of melanomas, which may lead to the resistance of melanomas tochemotherapy and molecular-target-therapy agents such as BRAFV600Einhibitors. Thisis frequently associated with downregulation or loss of PTEN which is a member ofinositol polyphosphate3-phosphatases. However, the role of inositol polyphosphate5-phosphatases which are similar to PTEN in carcinogenesis and progression ofmelanoma has not been delineated. Based on these, this study aims to examine thebiological role of phosphatidylinositol4,5-bisphosphate5-phosphatase (PIB5PA) incarcinogenesis and progression of melanoma and clarify the molecular mechanisms ofnegative regulation of PI3K/Akt pathway. It is expected to provide a new moleculartarget for individual therapy of melanoma. Furthermore, the exploration for epigeneticmechanisms of downregulation of PIB5PA in melanomas can provide a new idea forstudy of the mechanisms of carcinogenesis and progression of melanoma. Meanwhile, the identification of HDAC downregulating PIB5PA expression will be helpful todevelop more specific HDAC inhibitors that can inhibit melanoma growth.Methods (1) The expression of PIB5PA, PTEN and pSer473-Akt in melanoma tissuearrays, fresh melanoma isolates and melanoma cell lines were detected byimmunohistochemistry, western blot and qRT-PCR, respectively.(2) ME1007andMel-FH cells in which there are overly lowest expression of PIB5PA and highlyactivated Akt were transiently transfected with PIB5PA cDNA. In addition, ME1007and Mel-FH sublines (ME1007.PIB5PA and Mel-FH.PIB5PA) were established througha lentivirus-based inducible PIB5PA gene expression system in response to4-OHT. Theexpression of PIB5PA and pSer473-Akt, cell proliferation and apoptosis in transfectedcells or4-OHT-induced cells were determined by western blot, BrdU proliferationassays, clonogenic assays and FCM, respectively. The anchorage-independent growth ofmelanocytes which were transducted with PIB5PA and PTEN shRNA were detected bysoft agar clonogenic assays. Tumor growth of ME1007.PIB5PA cells subcutaneouslytransplanted into nu/nu mice treated with4-OHT or the vehicle control to determine thefunctional significance of expression of PIB5PA in melanoma growth in a melanomaxenograft mouse model. Co-transfection with myr-Akt or co-transduction with AktshRNA in the melanoma cells overexpressing or knockdown of PIB5PA were subjectedto detect its reversed effect of cell proliferation and tumor growth by use of western blot,BrdU proliferation assays, clonogenic assays, and melanoma xenograft mouse model.(3)The activation of Akt in melanoma cells overexpressed PIB5PA and/or PTEN whichwere serum-starved before stimulation with EGF were detected by western blot, and theThe anchorage-independent growth of melanocytes which were transducted withPIB5PA or PTEN or co-transducted with PIB5PA and PTEN shRNA were detected bysoft agar clonogenic assays.(4) The mutations of PIB5PA gene in10melanoma cellswere clarified by use of exon sequencing. Copy number variation of PIB5PA gene in10 melanoma cell lines and20fresh melanoma isolates were quantitated by qRT-PCRanalysis of genomic DNA. The mRNA and protein expression of PIB5PA in melanomacells treated with DNA methylation inhibitor5-aza and different specific HDACinhibitors were detected by qRT-PCR and western blot, respectively. The identificationof HDAC downregulating PIB5PA in melanoma cells was subjected to siRNAknockdown of specific HDAC in melanoma cells through detection of PIB5PAexpression by western blot. The further investigations of mechanisms ofhypoacetylation in downregulation of PIB5PA in melanomas were completed by use ofdual luciferase reporter gene assays and chromatin immunoprecipitation assays.Results (1) The expression of PIB5PA was frequently reduced or lost in melanomas andmelanoma cells compared with nevi and melanocytes, respectively. Moreover, theexpression of PIB5PA was positivly correlated with PTEN in melanomas and melanomacells.(2) Overexpression of PIB5PA, but not the mutant, caused a decreased inpSer473-Akt, inhibition of cell proliferation and a proportion of apoptosis in melanomacells. It could be reversed by co-transfection with myr-Akt. Knockdown of PIB5PA inhuman melanocytes activated Akt and resulted in anchorage-independent growth ofmelanocytes, and it could be reversed by co-transducted with Akt shRNA as well.Exogenous inducible expression of PIB5PA retarded melanoma growth in a xenograftmouse model in vivo, which could be reversed by co-transducted with myr-Akt.(3)When subsequently stimulated with EGF, the kinetics of Akt activation was deceleratedand the magnitude reduced in the melanoma cells overexpressing PIB5PA. Asanticipated, transfection of PTEN also resulted in inhibition of activation of Akt;however, the kinetics of Akt activation triggered by EGF in these transfected cells wassimilar to that of the control cells. The inhibitory effect on Akt activation was furtherenhanced when a PTEN construct was introduced into ME1007.PIB5PA andMel-FH.PIB5PA cells followed by treatment with4-OHT. Co-knockdown of PIB5PA and PTEN in Mel-AT and Mel-JD cells that expressed relatively high levels of theproteins resulted in further increases in activation of Akt in comparison withknockdown of PIB5PA or PTEN alone. Similarly, Co-knockdown of PIB5PA and PTENled to enhanced Akt activation and enlarged anchorage-independent colonies comparedwith knockdown of PIB5PA or PTEN individually in melanocytes. Neverthless, thenumbers of colonies formed by dual knockdown cells appeared fewer than those formedby PIB5PA or PTEN single knockdown cells, and the proliferation rate of the doubleknockdown cells was decreased.(4) Although gene copy number reduction contributedto the downregulation of PIB5PA in a proportion of melanomas (<20%), histonehypoacetylation was the main cause of suppression of PIB5PA in melanoma whichmediated by HDAC2and-3through binding to the transcriptional factor Sp1at thePIB5PA gene promoter region (-516/-116).Conclusion The activation of PI3K/Akt signaling pathway mediated by downregulationof PIB5PA and its cooperation with PTEN in melanoma may be important determinantof carcinogenesis and progression of melaonma and the resistance to chemotherapy.Histone hypoacetylation inhibits the expression of PIB5PA in melanoma, in thatHDAC2and-3are associated with PIB5PA gene promoter region responsive to theirinhibition through binding to the transcriptional factor Sp1.