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Sources of Funding: This work was supported by the Shaanxi Provincial Natural Science Foundation (2016JQ8011 to J. Zhou) and the National Natural Science Foundation of China (NSFC 81502205 to L. Liang, 81602237 to J. Zhou).

Introduction

Renal cell carcinoma (RCC) is the most lethal urologic malignancy, however, the molecular events involved in RCC pathogenesis and progression are still not well defined. DAB2IP, a novel member of Ras GTPase-activating protein gene family, is associated with cell proliferation, apoptosis, metastasis and chemo- or radio-resistance in several cancers. We have recently reported the tumor suppressive role of DAB2IP in RCC development, and identified one CpG methylation biomarker located at upstream of the transcription start site of DAB2IP that was associated with poor survival in three independent large-scale cohorts of RCC patients. In the present study, we determined molecular mechanisms of DAB2IP in suppressing the growth of RCC.

Methods

DAB2IP knockdown (KD) and overexpressing cells were generated by RNAi or cDNA transfection technologies. MTT, cell-cycle assays and xenograft models were performed to investigate the effect of DAB2IP on RCC cell growth. Mechanisms of DAB2IP in regulating cell growth were delineated by a variety of molecular biologic techniques such as real-time PCR, western blot, and immunoprecipitation. Relationships between DAB2IP, its downstream gene and tumor size were validated by clinical samples, and the associations of critical proteins and RCC patient survival were analyzed.

Results

DAB2IP KD cells exhibited promoted G1/S phase cell-cycle progression and enhanced cell proliferation, whereas, DAB2IP-overexpressing cells showed decreased growth ability. Mechanistically, DAB2IP promoted ubiquitination and degradation of a critical negative regulator of the cell-cycle, p27 (Kip1). Further signaling cascade profiling demonstrated that the Proline-rich domain in C terminal (CPR) of DAB2IP inhibited the activity of AKT phosphorylation (S473). Loss of DAB2IP, thus, promoted the phosphorylation of p27 on Serine 10 (S10) by AKT. Phosphorylated p27 was then sequestrated in cytosol, and subsequently ubiquitinated for degradation. Introduction of a constitutively activated AKT which was not affect by DAB2IP ablated the effect of DAB2IP on RCC growth, while introduction of a mutated p27 (p27 S10A) which escaped from the phosphorylation of AKT significantly restored the suppressive role of DAB2IP. In RCC patients, DAB2IP negatively correlated with tumor size and positively correlated with p27 expression. Survival analysis indicated that DAB2IP was associated with a better overall survival of RCC patient.

Conclusions

This study identifies DAB2IP as a tumor suppressor in RCC, and its mechanism of action is to stabilize p27 via suppressing PI3K/AKT signaling.

Funding

This work was supported by the Shaanxi Provincial Natural Science Foundation (2016JQ8011 to J. Zhou) and the National Natural Science Foundation of China (NSFC 81502205 to L. Liang, 81602237 to J. Zhou).