CLP36 Promotes p53-deficiency Induced Tumor Growth via Up-regulation of YAP1 Signaling

The tumor suppressor protein p53 is a critical protector of genome integrity in response to biological and environmental cues. Impaired or abnormally functioning p53 contributes to initiating tumorigenesis and promoting cancer progression. Although extensive research has been devoted to revealing p53 function and regulation in cancer progression, the molecular mechanism underlying p53 deficient tumor progression remains obscure due to the complexity of p53 mutations.

CLP36, also known as PDZ and LIM domain protein 1, is a ubiquitously expressed cytoskeletal protein. As a scaffold protein, CLP36 can recruit specific signal proteins to actin and organize signaling complexes at the cytoplasm.

Furthermore, CLP36 is abnormally expressed in various malignancies and closely associated with tumor growth and metastasis. To explore the role of CLP36 in p53 deficient cancer, I examined the expression of CLP36 in p53 knockout mouse tissue and found that CLP36 is upregulated in p53 null mouse tissue. Then, with in vivo and in vitro approaches, I provided a series of evidence showing CLP36 functions as an oncoprotein in p53 deficient malignancies. Functional studies indicate that knockdown of CLP36 significantly diminished the oncogenic abilities of p53 null sarcoma cells. Also, ablation of CLP36 in p53 knockout mice confers significant protection from spontaneous tumorigenesis.

Considering the potential diagnostic and therapeutic values of CLP36 in p53 null tumor, I sought to further reveal the molecular mechanism achieving its functions. Recently, crosstalk between YAP1 function and p53 status in cancer was revealed. Loss of p53 function in cancers may be accompanied by uncontrolled YAP1 oncogenic activities. To verify whether CLP36 executes its function in p53 deficient cancer through YAP1, I detected the alterations of the levels of YAP1 after manipulation of CLP36 expression. Our results indicated that deletion of CLP36 significantly decreased the protein levels of YAP1, while re-expressing CLP36 rescued the YAP1 protein levels in CLP36 deficient cells. Since YAP1 degradation is tightly governed by phosphorylation through multiple signaling pathways, I first detected the alterations of the phosphorylation levels of YAP1 upon CLP36 inhibition. Phosphorylation of YAP1 is not significantly changed in CLP36 deficient cells. Moreover, it was reported that YAP1 degradation is

regulated by E3 ubiquitin ligases, which catalyzes YAP1 ubiquitination and ultimately leading to degradation via a proteosome-dependent manner. Treatment of proteosome inhibitor, MG132, could accumulate YAP1 in CLP36 knockdown cells, indicating that CLP36 may stabilize YAP1 predominantly via regulation of its proteasome-mediated degradation. Furthermore, I found that inhibition of E3 ligase AIP4 significantly reversed the downregulation of YAP1 levels induced by the deletion of CLP36. Mechanistically, the CLP36 function is dependent on its PDZ and ZM domain, which is essential for interacting with α-actinin. Truncated CLP36 inhibiting the α-actinin-binding activity abolishes the ability of CLP36 to stabilize YAP1 and promote oncogenic properties.

In this study, I identified the promotional role of CLP36 in p53-null tumor growth via regulating YAP1 degradation. Importantly, I proposed a new molecular regulation between CLP36 and YAP1 in p53 deficient cancer progression and demonstrated a potential diagnostic and therapeutic strategy for cancers harboring loss-of-function p53 mutation.

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