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YAP Subcellular Localization and Hippo Pathway Transcriptome Analysis in Pediatric Hepatocellular Carcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Pediatric hepatocellular carcinoma (HCC) is a rare tumor which is associated with an extremely high mortality rate due to lack of effective chemotherapy. Recently, the Hippo pathway and its transcriptional co-activator Yes-associated protein (YAP) have been shown to play a role in hepatocyte proliferation and development of HCC in animal models. Therefore, we sought to examine the activity of YAP and the expression of Hippo pathway components in tumor and non-neoplastic liver tissue from 7 pediatric patients with moderately differentiated HCC. None of the patients had underlying cirrhosis or viral hepatitis, which is commonly seen in adults with HCC. This highlights a major difference in the pathogenesis of HCC between children and adults. We found a statistically significant increase in YAP nuclear localization in 100% of tumors. YAP target gene (CCNE1, CTGF, Cyr61) mRNA expression was also increased in the tumors that had the most significant increase in YAP nuclear localization. Based on Ki67 co-localization studies YAP nuclear localization was not simply a marker of proliferation. Our results demonstrate a clear increase in YAP activity in moderately differentiated pediatric HCC, providing evidence that it may play an important role in tumor survival and propagation.

No MeSH data available.


YAP1 mRNA transcript and YAP protein levels.(a) Substantial increases in transcription were seen only in Patients 3, 4, and 5. The remaining patients had minimal change. (b) Western blot demonstrating YAP protein levels. (c) Bar graph quantification of immunoblot in (panel b).
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f6: YAP1 mRNA transcript and YAP protein levels.(a) Substantial increases in transcription were seen only in Patients 3, 4, and 5. The remaining patients had minimal change. (b) Western blot demonstrating YAP protein levels. (c) Bar graph quantification of immunoblot in (panel b).

Mentions: Given the increased nuclear localization of YAP in tumor cells we sought to determine whether this was associated with an increase in YAP1 transcription and protein expression. Total RNA was extracted from tumor and non-neoplastic liver samples from the same patient, and analyzed using qPCR following reverse transcription (qRT-PCR). While YAP1 expression was upregulated by greater than 1.5 fold in Patients 3, 4, 5, and 7, there was not a significant change in YAP1 mRNA levels between non-neoplastic liver and tumor as a cohort (Fig. 6a). When we compared FLM-HCC to non-FLM-HCC, there was a greater overall increase in YAP1 expression in the non-FLM-HCC group; however it did not reach statistical significance. Whole cell lysate from each sample was analyzed using Western blotting (Fig. 6b,c). Only Patient 4 showed a substantial increase in YAP protein levels, correlating with the large increase this patient had in YAP1 expression. Interestingly, although Patient 3 also had a large increase in YAP1 expression, YAP protein levels for this patient did not change substantially. Patient 2 had a decrease in YAP protein levels, correlating with a decrease in YAP1 expression. However, Patient 6, who also had decreased YAP1 expression, had no substantial change in YAP protein levels. The remaining patients also had minimal change in YAP levels. There was no significant change in YAP protein expression in the cohort overall, or when the tumors were sub-analyzed based on fibrolamellar histology.


YAP Subcellular Localization and Hippo Pathway Transcriptome Analysis in Pediatric Hepatocellular Carcinoma
YAP1 mRNA transcript and YAP protein levels.(a) Substantial increases in transcription were seen only in Patients 3, 4, and 5. The remaining patients had minimal change. (b) Western blot demonstrating YAP protein levels. (c) Bar graph quantification of immunoblot in (panel b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015017&req=5

f6: YAP1 mRNA transcript and YAP protein levels.(a) Substantial increases in transcription were seen only in Patients 3, 4, and 5. The remaining patients had minimal change. (b) Western blot demonstrating YAP protein levels. (c) Bar graph quantification of immunoblot in (panel b).
Mentions: Given the increased nuclear localization of YAP in tumor cells we sought to determine whether this was associated with an increase in YAP1 transcription and protein expression. Total RNA was extracted from tumor and non-neoplastic liver samples from the same patient, and analyzed using qPCR following reverse transcription (qRT-PCR). While YAP1 expression was upregulated by greater than 1.5 fold in Patients 3, 4, 5, and 7, there was not a significant change in YAP1 mRNA levels between non-neoplastic liver and tumor as a cohort (Fig. 6a). When we compared FLM-HCC to non-FLM-HCC, there was a greater overall increase in YAP1 expression in the non-FLM-HCC group; however it did not reach statistical significance. Whole cell lysate from each sample was analyzed using Western blotting (Fig. 6b,c). Only Patient 4 showed a substantial increase in YAP protein levels, correlating with the large increase this patient had in YAP1 expression. Interestingly, although Patient 3 also had a large increase in YAP1 expression, YAP protein levels for this patient did not change substantially. Patient 2 had a decrease in YAP protein levels, correlating with a decrease in YAP1 expression. However, Patient 6, who also had decreased YAP1 expression, had no substantial change in YAP protein levels. The remaining patients also had minimal change in YAP levels. There was no significant change in YAP protein expression in the cohort overall, or when the tumors were sub-analyzed based on fibrolamellar histology.

View Article: PubMed Central - PubMed

ABSTRACT

Pediatric hepatocellular carcinoma (HCC) is a rare tumor which is associated with an extremely high mortality rate due to lack of effective chemotherapy. Recently, the Hippo pathway and its transcriptional co-activator Yes-associated protein (YAP) have been shown to play a role in hepatocyte proliferation and development of HCC in animal models. Therefore, we sought to examine the activity of YAP and the expression of Hippo pathway components in tumor and non-neoplastic liver tissue from 7 pediatric patients with moderately differentiated HCC. None of the patients had underlying cirrhosis or viral hepatitis, which is commonly seen in adults with HCC. This highlights a major difference in the pathogenesis of HCC between children and adults. We found a statistically significant increase in YAP nuclear localization in 100% of tumors. YAP target gene (CCNE1, CTGF, Cyr61) mRNA expression was also increased in the tumors that had the most significant increase in YAP nuclear localization. Based on Ki67 co-localization studies YAP nuclear localization was not simply a marker of proliferation. Our results demonstrate a clear increase in YAP activity in moderately differentiated pediatric HCC, providing evidence that it may play an important role in tumor survival and propagation.

No MeSH data available.