The RNA helicase DHX33 has been found to be overexpressed in human cancers, where it promotes cancer development. reduction. These data support the notion that disruption of DHX33 function could be an important application for cancer therapy. release (16). BH3-only proteins, such as BIM and PUMA, directly activate BAX, which can be reversibly inhibited by prosurvival proteins, such PF-04880594 as Bcl-2, Bcl-xl, and Bcl-w (15, 16). The other BH3-only protein, BAD, indirectly activates BAK or BAX through competitively inhibiting Bcl-2 (17). In human cancers, Bcl-2 is frequently overexpressed (18). Although the relationship of Bcl-2 family members is well characterized, the upstream regulatory pathway driving the PF-04880594 expression of Bcl-2 family proteins remains incompletely understood. We and others have previously found that knockdown of DHX33 leads to apoptosis in human cancer cells (8, 11). However, the underlying mechanism of this process remains obscure. In this study, we reveal that DHX33 represses apoptosis through the direct upregulation of gene transcription. We identify that AP-2 is a binding partner for DHX33 and that DHX33 acts as a coactivator for AP-2 to promote the transcription of antiapoptotic gene. In addition, we found that normal human mammary and lung epithelial cells are less sensitive to DHX33 deficiency, indicating a unique and heightened sensitivity to DHX33 expression in cancer cells but not normal cells. Together, our data implicate the therapeutic potential of DHX33 in cancer treatment. RESULTS DHX33 supports breast cancer cell survival. We have previously observed that lung cancer cells rapidly undergo cell apoptosis after DHX33 knockdown. To investigate whether DHX33 promotes cell survival in other cancer cell types, we analyzed the effect of DHX33 knockdown in breast cancer cell lines. We applied several different shRNAs targeting DHX33 in BT549, HCC1806, and SKBR3 cells, respectively, with shScramble as a control. As shown in Fig. 1A and ?andD,D, these shRNAs efficiently reduced DHX33 protein levels. DHX33 deficiency triggered cell death, as visualized by light microscopy of enhanced refractive cells (Fig. 1B and ?andE).E). Through annexin V staining, we determined that these DHX33-deficient cells underwent apoptosis (Fig. 1C). To evaluate the effect of DHX33 knockdown in breast cancer cells family gene expression. To investigate the underlying mechanism for apoptosis induced by DHX33 reduction, we first analyzed the changes in total gene expression after DHX33 knockdown. As shown by RNA sequencing (RNA-seq) results in H1299 lung cancer cells (Fig. 3A), we found that genes involved in the mitochondrial pathway of apoptosis were highly deregulated. The mRNA levels of several Bcl-2 family members demonstrated altered expression after DHX33 knockdown. The gene itself was significantly downregulated whereas BAD, BIM, BMF, and PUMA genes were upregulated in H1299 cells after DHX33 knockdown. To check whether these results also occurred in other cell types, we further performed reverse transcription-PCR (RT-PCR) analysis for MDA-MB231 cells, BT549 cells, and MCF10A cells. As shown in Fig. 3B to ?toD,D, after DHX33 knockdown, Bcl-2 was downregulated, BAD and BIM were upregulated in all three cell lines, whereas BMF, BAK, BOK, and BAX were upregulated in a cell-type-dependent manner. Interestingly, we observed that Bcl-xl, Bcl-w, and MCL1 were upregulated in different cell lines after DHX33 knockdown, implicating a possible feedback regulatory mechanism. To confirm the results from RNA-seq, we further performed immunoblot analysis for both lung cancer and PF-04880594 breast cancer cells (Fig. 4A and ?andB).B). Depending on the different cell lines, DHX33 knockdown dramatically altered the expression of NAV3 at least one or multiple Bcl-2 family members, particularly gene expression. The altered expression of Bcl-2 family members caused pre-caspase 7 to be cleaved into caspase 7 (Fig. 4A and ?andB)B) and PARP was also cleaved after DHX33 knockdown (Fig. 4A and ?andB).B). To confirm the expression changes of Bcl-2 family members at the transcriptional level, RT-PCR analysis was further performed after DHX33 knockdown. As shown in Fig. 4C, we found that after DHX33 knockdown, gene transcription was downregulated in multiple human cancer cell lines, while the transcript PF-04880594 levels of BAD, BIM, and BMF were elevated only in certain cell types, such as H1299 cells, but not in MDA-MB-231 cells. Deregulation of these important genes should cause oligomerization of BAX/BAK protein on the outer membranes of mitochondria, which in turn leads to mitochondrion-mediated apoptosis. We therefore analyzed the membrane potential of mitochondria PF-04880594 after DHX33 knockdown in cancer cells with JC-1 staining. Under normal conditions, JC-1 will polymerize in the mitochondria, emitting red fluorescence. However, in apoptotic cells, due.