br Introduction It is well known that when epithelial

Introduction It is well known that when epithelial cells are deprived of anchorage, they undergo detachment-induced apoptosis, also known as anoikis. Cancer cells, by contrast, are able to suppress anoikis and survive under anchorage -independent conditions in the circulating system, which is thought to be a critical step for cancer cells to form a distant metastatic lesion [1]. Hepatocellular carcinoma (HCC) is an aggressive cancer with poor prognosis due to the high frequency of tumor metastasis [2]. Our previous study showed that HCC cells acquired anoikis-resistance and maintained survival after anchorage deprival [3], whereas the molecular mechanisms underlying this process have yet to be fully understood. In the current study, we showed that tripartite motif-containing protein (TRIM) 31, a new member of the TRIM family, was involved in the anoikis-resistant process of HCC cells. TRIM family members are characterized by presence of the common N-terminus composed of a RING-finger, one or two B-box domains and a coiled-coil domain. TRIM family proteins are involved in a broad range of biological processes and their alterations result in diverse pathological conditions such as genetic diseases, viral infection and cancer development [4]. Whether TRIM family members play a role in regulation of resistance to anoikis of HCC cells has never been defined.
Materials and methods
Results
Discussion It is well known that p53 plays a central role in coordinating the responses to stresses induced by a wide array of stimuli. Under normal conditions, cellular p53 protein levels are maintained at basal level. However, in response to genotoxic related stresses, p53 protein level is increased which further triggers either rta 3 arrest or apoptosis of the host cells [12], [13]. Recent reports suggested potential role of p53 in regulation of anoikis in several types of malignancies, including lung cancer, squamous cell carcinoma, and breast cancer [14], [15], [16], whereas its exact function and molecular mechanism is far from being clarified. Whether dysregulation of p53 is involved in regulation of anoikis of HCC cells has never been reported. In this study, we revealed that TRIM31 promoted anoikis-resistance of HCC cells by directly targeting p53 for ubiquitous degradation, and loss of p53 further abolished detachment-induced apoptosis. Ubiquitination is an enzymatic post-translational modification regulating diverse cellular processes. The K48-linked poly-ubiquitination is a canonical signal for proteasomal degradation, whereas the K63-linked poly-ubiquitination mediates proteasome-independent cellular processes such as DNA repair and signaling protein complex assembly [17], [18]. In the ubiquitin conjugation system, E3 ligases function as the specific recognition modules and interact with both E2 and substrate. Many TRIM family members contain RING domain, which confers E3 ligase activity to these TRIM proteins [19]. In this study, we demonstrated for the first time that TRIM31 tagged K48-linked poly-ubiquitin chains to p53 and led to the ubiquitous degradation of p53 via the RING domain. Moreover, we further demonstrated that lack of p53 expression in the anchorage-deprived HCC cells was responsible for AMPK pathway activation. AMPK pathway is activated under stresses of an increased adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio. It has been described as “fuel gauge of the cell” and is responsible for mediating cellular responses to energy stress [20]. AMPK is phosphorylated at Thr172 within its α subunit and this phosphorylation is essential for its functional activation [21]. Though activation of AMPK pathway was reported to inhibit disease in some types of cancer [22], [23], it was reported to promote caner progression in other types of cancer [24], [25], [26]. Recently, activation of AMPK pathway has been implicated in anoikis-resistance of cancer cells [27], [28], however the role of AMPK pathway activation upon matrix detachment of HCC cells has not been clarified. In this study, we showed that overactivation of AMPK pathway was responsible for anoikis-resistance and enhanced malignancy of anchorage-deprived HCC cells. It is reported that autophagy triggered by loss of p53 is closely associated with AMPK activation [29], [30]; however, the involvement of p53-AMPK axis in the regulation of anoikis is investigated here for the first time. We demonstrated that TRIM31-mediated p53 degradation was responsible for AMPK pathway activation in the anchorage-deprived HCC cells. Thus our study revealed that when anchorage was deprived in HCC cells, TRIM31 was upregulated in response to this extracellular stress and it further targeted p53 for K48-linked ubiquitous degradation. Loss of p53 led to overactivation of AMPK pathway, which further mediated cellular response to anchorage-deprival induced energy crisis, and helped non-anchored HCC cells to survive in circulation and to further form a metastatic lesion.