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Homeodomain-interacting protein kinase-2 (HIPK2) can either promote or inhibit transcription depending on cellular context. In this study, we show that a new HIPK2 isoform increases TEAD reporter activity in NSCLC cells. We detected HIPK2 copy number gain in 5/6 (83.3%) NSCLC cell lines. In NSCLC patients with high HIPK2 mRNA expression in the Human Protein Atlas, the five-year survival rate is significantly lower than in patients with low expression (38% vs 47%; p = 0.047). We also found that 70/78 (89.7%) of NSCLC tissues have moderate to strong expression of the N-terminal HIPK2 protein. We detected and cloned a novel HIPK2 isoform 3 and found that its forced overexpression promotes TEAD reporter activity in NSCLC cells. Expressing HIPK2 isoform 3_K228A kinase-dead plasmid failed to increase TEAD reporter activity in NSCLC cells. Next, we showed that two siRNAs targeting HIPK2 decreased HIPK2 isoform 3 and YAP protein levels in NSCLC cells. Degradation of the YAP protein was accelerated after HIPK2 knockdown in NSCLC cells. Inhibition of HIPK2 isoform 3 decreased the mRNA expression of YAP downstream gene CTGF. The specific HIPK2 kinase inhibitor TBID decreased TEAD reporter activity, reduced cancer side populations, and inhibited tumorsphere formation of NSCLC cells. In summary, this study indicates that HIPK2 isoform 3, the main HIPK2 isoform expressed in NSCLC, promotes YAP/TEAD transcriptional activity in NSCLC cells. Our results suggest that HIPK2 isoform 3 may be a potential therapeutic target for NSCLC.

Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly (n = 142, r = 0.514, P < 0.001). Second, in cell lines that express high levels of PD-L1 (H460, SKLU-1, and H1299), the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1 (A549, H2030, and PC9) (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3'UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.

A small population of cancer cells called cancer-initiating cells or cancer stem cells (CSCs) are involved in drug resistance, metastasis, and cancer relapse. Finding pathways that regulate CSC is very important for clinical therapy. ATP-binding cassette sub-family G member 2 (ABCG2) plays a role in side population (SP) cell formation and contributes to chemotherapy resistance in common forms of cancer. Yes-associated protein 1 (YAP1) is a major transcriptional effector of the Hippo pathway, which plays important roles in organ size control and tumorigenesis. In this study, we found ABCG2 and YAP1 were both overexpressed in lung cancer SP cells. Disruption of YAP1 expression by siRNA attenuated the expression of ABCG2 transcript and significantly reduced the percentage of SP cells and sphere formation in lung cancer cells. Overexpression of YAP1 in lung cancers led to an increase in ABCG2 expression and increased the percentage of SP cells. However, overexpression of YAP1 in purified non-SP cells did not increase ABCG2 expression and the percentage of SP cells, which may be due to the inhibition of YAP activity through phosphorylation. YAP1 directly transcriptionally regulated ABCG2 by binding to the promoter of ABCG2. Moreover, the YAP1 inhibitor verteporfin and YAP1 siRNA downregulated ABCG2 level through inhibition of YAP1 in lung cancer cells and sensitized them to the chemotherapy drug doxorubicin. Our study adds a new function for YAP1 that may be relevant to drug resistance and cancer therapy through regulation of ABCG2 and side population cell formation in lung cancer.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P