Download Alterations of the EGFR and Hippo/ Yes

Alterations of the EGFR and Hippo/ Yes-associated protein (YAP) pathway have been found in
non-small cell lung cancer (NSCLC).
In part 1, we have investigated the connection between ERK1/2 and Hippo/YAP pathway
in NSCLC. Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in
human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors
decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA
levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP
protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level
was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein
level and Hippo reporter activity after siRNA knockdown targeting 3'UTR of the ERK2 gene in
NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells.
Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either
one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and
transcriptional activity of the Hippo pathway in NSCLC cell lines. Our results suggest that
ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates
expression of the downstream genes of the Hippo pathway to suppress migration and invasion
of NSCLC cells.
In part 2, we have shown that YAP Promotes Erlotinib Resistance in Human NSCLC Cells.
YAP is a main mediator of the Hippo pathway, which promotes cancer development. Here we
show that YAP promotes resistance to erlotinib in human 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<0.05), as
did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA
expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental
HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of
erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter
activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05).
Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of
H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration,
invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP
promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of
YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.
In part 3, we have shown that YAP regulates cancer stem cell-like properties via ABCG2
in human lung cancer cells. A small population of cancer cells called cancer initiating cells or
cancer stems cells (CSCs) involved in drug resistance, metastasis, and cancer relapse. Finding
pathways regulating CSCs properties is very important for clinical therapy. ATP-binding cassette
sub-family G member 2 (ABCG2) plays a role in the side population (SP) cell formation and
contributes to chemotherapy resistance in common forms of cancers. We asked whether YAP
regulates ABCG2 in lung cancer cells. In this study, we found ABCG2 and YAP were both
overexpressed in lung cancer SP cells. Disruption of YAP expression by siRNA attenuated the
expression of ABCG2 transcript and protein and significantly reduced the SP fraction in lung
cancer cells. Overexpression of YAP in lung cancers led to an increase in ABCG2 expression
and increased SP fraction. YAP directly transcriptionally regulated ABCG2 by co-binding to the
promoter of ABCG2 (with TEAD). Moreover, the YAP inhibitor vertepofin downregulated ABCG2
level through inhibition of YAP in lung cancer cells and sensitized them to the chemotherapy
drug doxorubicin. Our study adds new function for YAP that may be relevant to drug resistance
and cancer therapy through regulation of ABCG2.
In part 4, we have shown that YAP is an oncogenic driver and a key therapeutic target of
mesothelioma. Malignant pleural mesothelioma (mesothelioma) is a very aggressive form of
cancer that resistant to current therapy. The poor prognosis of this disease has been associated
with elevated Yes-associated protein (YAP) activity due to mutations of Hippo pathway
components. Here, we show that specific targeting of YAP has a remarkable effect on the Hippo
pathway and tumor activity of human mesothelioma cells. Firstly, overexpression of YAP was
detected in 62.5% (40/64) of clinical samples with nuclear staining by immunohistochemistry
and in 4/6 mesothelioma cell lines by real-time PCR. TEAD4, which belongs to TEAD family of
transcription factors and mediates the biological function of YAP, was also overexpressed in
54.7% (35/64) of these tissues and in the same 4/6 mesothelioma cell lines. Secondly, silencing
of YAP by siRNAs down-regulated the reporter activity of the Hippo pathway, whereas forced
over-expression of the YAP gene rescued the Hippo reporter activity after siRNA knockdown
targeting 3’UTR of the YAP gene in mesothelioma cells. Thirdly, the YAP inhibitor verteporfin,
which disrupts the formation of the YAP-TEAD complex, also down-regulated transcriptional
activity of the Hippo pathway in mesothelioma cell lines. Fourthly, verteporfin treatment showed
high sensitivity to decrease the cell viability of mesothelioma cell lines. Finally, inhibition of YAP
by verteporfin treatment reduced the migration, invasion and self-renewal of cancer stem cells in
mesothelioma cells. Our results collectively suggest that YAP is an oncogenic driver and a key
therapeutic target of mesothelioma.
In part 5, we have recently been investigating the role of Hippo/YAP signaling in
metastasis of lung adenocarcinoma and mesothelioma in several animal models. In
addition, we are currently investigating and developing small molecule inhibitors targeting
Hippo/YAP pathway for targeted therapy of lung cancer and mesothelioma.