Reduced expression of myosin-9 and repressed phosphor- ylation of AKT and GSK-3β

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 elicitingEMTandcellproliferationandmigrationconvergeonAKTand 3.5. MICAL2 nuclear export depended on myosin-9 in LUAD cells myosin-9. Reduced expression of myosin-9 and repressed phosphor- ylation of AKT and GSK-3β in the MICAL2-knockdown PC-9 were To further investigate the factors mediating the nucleocytoplasmic confirmed, indicating the potential role of these signaling pathways in shuttling of MICAL2 cck-8 structure, immunoprecipitation (using anti-MICAL2 anti- mediating MICAL2 function in LUADs (Fig. 2G). body) coupled with mass spectrometry (IP-MS) was applied to search To further verify the tumor promoting role of MICAL2 in LUADs, a for the endogenous MICAL2-binding proteins in PC-9 cells. After sub- lentiviral gene delivery system was used to overexpress MICAL2 in the tracting the non-specific binding proteins using isotype-controlled IP- MICAL2-low A549 cells with undetectable levels of endogenous MS, 108 proteins were identified as MICAL2-binding partners with in- MICAL2 expression (Fig. 2A and 2B). CCK8 and plate clone assays il- tensity ratios>10 (MICAL2-Ab: IgG > 10, data not shown). The FDR lustrated that MICAL2 promoted A549 cell proliferation (Fig. 2C and was analyzed byperforminga concatenated decoy database search and 2D). Transwell assays further revealed that MICAL2 enhanced the mi- the identified proteins were reported at FDR<1%. Myosin-9 gration and invasion abilities of A549 cells (Fig. 2E). MICAL2-over- (MYH9)—a motor protein associated with certain nucleocytoplasmic expressing A549 cells also exhibited mesenchymal-like phenotype with shuttling proteins [30–34]—was among the top-five MICAL2-binding longer and more numerous protuberances (Figs. 2F, 2C, S1). Accord- proteins ranked by intensity ratio (Fig. 4A, Table S1). We further per- ingly, western blotting confirmed the increased expression of the EMT formed immunoprecipitation (using anti-myosin-9 antibody) coupled markers vimentin and β-catenin and the decreased expression of ZO-1 with a parallel reaction monitoring assay (IP-PRM) and confirmed en- and E-cadherin (Fig. 2G). Interestingly, myosin-9 expression and dogenous MICAL2-myosin-9 interaction in the PC-9 cells (Fig. 4B). In phosphorylation ofAKTandGSK-3βweresignificantlyincreasedinthe addition, exogenous MICAL2 and myosin-9 binding was identified by a MICAL2-overexpressing A549 cells (Fig. 2G). Altogether, these data co-IPassayinHEK293Tcells(Fig.4C).Theco-IPassayalsoshowedthat suggest that MICAL2 promotes tumor malignancy and induces EMT in si-myosin-9 negatively affected myosin-9 expression levels as well as LUADcellsalongwithincreasedlevelsofmyosin-9expressionandAKT binding capacity to MICAL2 (Fig. 4C). Next, to determine whether phosphorylation. myosin-9 affects the subcellular location of MICAL2 in LUAD cells, A549-MICAL2 cells were treated with ML7—a myosin-9 specific in- hibitor—and si-myosin-9. Immunofluorescence assays revealed that 3.4. MICAL2 was sensitive to LMB and relied on its C-terminal end to bothML7andsi-myosin-9treatmentledtothenuclearaccumulationof shuttle between the cytoplasm and the nucleus MICAL2(Fig.4D).Altogether,thesedatasuggestthatmyosin-9bindsto MICAL2 and promotes MICAL2 nuclear export. TofurtherunderstandhowMICAL2worksinLUADs,thesubcellular location of MICAL2 was investigated. These localizations have been 3.6. The cytoplasmic translocation of MICAL2 was required for its cancer- reporteddisparatelyfordifferentcelllines[14,15]orevenforthesame promoting activity in vitro and in vivo cell lines in different articles [16,19]. MICAL2 expression patterns variedinLUADs(Fig.1),withasimilarsituationobservedinLUADcell ThedatadescribedaboverevealedthatMICAL2isatumorpromoter lines (data not shown). This raises the question whether MICAL2 ac- in LUADs as well as a nucleocytoplasmic shuttling protein that is sen- tivelyshuttlesbetweenthecytoplasmandnucleusinLUADcells.Given sitivetoLMB,ML7,andsi-myosin-9treatments.Moreover,theMICAL2 that proteins larg###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-5.png####er than 40–60 kDa (full length of MICAL2, 127 kDa) C-terminal fragment contains the candidate NES sequence(s) for nu- barelydiffusefreelythroughnuclearporesandthattheMICAL2protein clear export. To investigate whether the cellular location of MICAL2 contains a nuclear localization sequence (NLS) [28], we sought to de- determines its cancer-promoting activity in LUAD cells RGX-104, we examined termine whethertheblockage ofactive nuclear exportby leptomycin B how LMB, ML7 and si-myosin-9 treatments and the deletion of the C- (LMB)—an inhibitor of export receptor chromosome maintenance re- terminal fragment affect MICAL2 tumor-promoting activities in LUAD. gion 1 [29]—affects MICAL2 cellular translocation. 

The immuno- A lentiviral approach was used to generate the A549 with −ΔC fluorescence assay followed by confocal imaging confirmed that LMB overexpression of wild type MICAL2 (MICAL2-WT), MICAL2 ,or −ΔC blocked the nuclear export of endogenous MICAL2, leading to nuclear control (Ctrl, Fig. 5A). Both MICAL2-WT and MICAL2 induced accumulation in the PC-9 cells (Fig. 3A). A similar observation was myosin-9 upregulation (Fig. 5A). The CCK8 assays revealed that MI- −ΔC madeforectopically overexpressedMICAL2intheA549cells(Fig.3B). CAL2 exhibits only minimal tumor-promoting activity that is still This indicated that MICAL2 is a nucleocytoplasmic shuttling protein, significantly higher than that of the Ctrl but much lower than that of −ΔC dependent on the exportin 1 nuclear export receptor. wildtypeMICAL2(Fig.5B).Incontrast,MICAL2 completelylostits MICAL2 contains a kinase domain (the MO domain), an actin- capacity to promote cell migration and invasion (Fig. 5C). This lack of binding domain (the CH domain), a previously identified NLS, and a migration/invasion-promoting activity is likely due to the mislocation −ΔC LIM domain [9,16]. Given that NLSs and NESs (nuclear export signals) but not misfolding of the MICAL2 protein, given that this protein are generally required for the active nuclear-cytoplasmic trafficking of maintained its ability to induce myosin-9 expression as well as weakly proteins [28], we attempted to allocate the NESs within MICAL2. We promoted cell proliferation.

To investigate the effect of MICAL2 and its 6 made several truncation mutants of Flag-tagged MICAL2 including nuclearexportonthemetastasispotentialofLUADcellsinvivo KX2-391,1×10 −ΔC −ΔNΔC −ΔN −ΔC MICAL2 , MICAL2 , and MICAL2 (Fig. 3C) and transfected A549-CTRL, A549-MICAL2 , or A549-MICAL2 cells were injected them into HeLa cells. Then immunofluorescence was performed using into the tail veins of nude mice. H&E staining showed increased me- anti-Flag antibody to detect the subcellular location of MICAL2 mu- tastatic nodule numbers in A549-MICAL2 cells but not A549-MI- −ΔN −ΔC tants. As shown inFig. 3C, whileMICAL2 with thedeletion of both CAL2 compared with A549-CTRL cells (Fig. 5D). the MO and CH domains exhibited a dispersed distribution throughout Finally, blocking MICAL2 cytoplasmic translocation using LMB, −ΔC the cells similar to wild type MICAL2 (MICAL2-WT), MICAL2 and ML7, or si-myosin-9 robustly blocked MICAL2 capacity to drive cell −ΔNΔC MICAL2 predominantly localized in the nucleus. Furthermore, proliferation, motility, and invasion (Fig. 6). This further supports the there was no statistical difference in the cytoplasmic positive rate be- premise that cytoplasmic MICAL2 exerts LUAD-promoting activity, −ΔC −ΔNΔC tween MICAL2 and MICAL2 , suggesting that the C-terminal which may functionally explain the different cellular locations of fragment of MICAL2 contains the NESs for actively exporting MICAL2. MICAL2 in LUADs and normal lung tissues. −ΔC Moreover, MICAL2 without the loss of the MO domain (kinase do- main) and CH domain (actin-binding domain) provides a useful fra- 3.7. Expression of MICAL2 and myosin-9 in LUAD tissues mework to investigate the functional contribution of MICAL2 in the nucleus. In order to confirm the correlation between MICAL2 and myosin-9 80