1Department of Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

2Department of Oncology, Ren ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127 China

3Operating Room, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

4Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

1Department of Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

1Department of Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

1Department of Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

1Department of Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China

The datasets used and/or analyzed, and materials used during the current study are available from the corresponding author on reasonable request.

B cell receptor associated protein 31 (BAP31) is closely associated with tumor progression, while the role and mechanism of BAP31 in gastric cancer (GC) remains unknown. This study explored that BAP31 was upregulated in GC tissues and high expression indicated poor survival of GC patients. BAP31 knockdown inhibited cell growth and induced G1/S arrest. Moreover, BAP31 attenuation increased the lipid peroxidation level of the membrane and facilitated cellular ferroptosis. Mechanistically, BAP31 regulated cell proliferation and ferroptosis by directly binding to VDAC1 and affected VDAC1 oligomerization and polyubiquitination. HNF4A was bound to BAP31 at the promoter and increased its transcription. Furthermore, knockdown of BAP31 inclined to make GC cells vulnerable to 5-FU and ferroptosis inducer, erastin, in vivo and in vitro. Our work suggests that BAP31 may serve as prognostic factor for gastric cancer and act as potential therapeutic strategy for gastric cancer.

Globally, gastric cancer (GC) severely affects human health and living quality, with high incidence rate and mortality rate [1, 2]. Despite the decreasing incidence of GC, recent statistical data indicate an increasing occurance in young population, which may change the age spectrum of GC patients [3]. Since GC is a heterogeneous disease both molecularly and phenotypically [4], an enormous potential exists for its treatment by using target therapy in combination with sequential lines of chemotherapy.

The B Cell Receptor Associated Protein 31 (BAP31) gene is located on X chromosome at q28, encoding a ubiquitously expressed membrane protein in endoplasmic reticulum (ER) [5]. As a chaperone protein in ER, BAP31 plays an important role in diverse biological processes such as the transport of membrane proteins from ER, and mediates crosstalk with mitochondria in caspase 8-mediated apoptosis, mitochondria dysfunction, etc [6–9]. BAP31 is upregulated in lung cancer, breast cancer, liver cancer, kidney cancer and many other kinds of cancer [10]. Furthermore, BAP31 may serve as a potential biomarker for prognostic prediction in non-small cell lung carcinoma and hepatocellular carcinoma [11, 12]. Also, it has been found that BAP31 exerts a significant influence on tumor proliferation in cervical cancer [13]. Similar phenotypes appeared at human embryonic stem cells, in which BAP31 attenuation induced cell apoptosis and inhibited cell proliferation [14]. BAP31 physically associated with epithelial cell adhesion molecule (EpCAM), and EpCAM expression significantly decreased with BAP31 knockdown, while BAP31 overexpression increased its level and enhanced cell adhesion [15]. Despite these progresses made in understanding the role played by BAP31 in cancers, research pertaining to unravel the role of BAP31 in GC is scarce.

As a novel form of regulated cell death, ferroptosis is characterized by iron overload, lipid reactive oxygen species (ROS) accumulation, and lipid peroxidation [16]. Increasing evidences have shown that ferroptosis extensively participates in physiological processes and various diseases, especially in cancer initiation, progression, and suppression [17, 18]. Examining the potentials of ferroptosis in cancer therapy is a fast-growing field of study, where several ferroptosis inducers have been found to be beneficial and promising in pre-clinical studies [19]. Currently, fluoropyrimidine with platinum-based chemotherapy serve as the standard regimen in advanced GC, in which drug resistance occurs frequently [20]. Cancer cells exhibit an increased iron demand compared with normal cells, and the iron dependency can make cancer cells more vulnerable to ferroptosis and creates the potential of ferroptosis to be a new promising way to kill therapy-resistant cancers [21]. Intriguingly, increasing evidence has emerged that ferroptosis inducer may help sensitize chemotherapy in gastric cancer. Zhang et al. demonstrated that CAFs secreted exosomal miR-522 to inhibit ferroptosis in cancer cells by targeting ALOX15 and blocking lipid-ROS accumulation, and blocking this axis enhanced sensitivity of chemotherapy [22]. There were studies demonstrating that inhibition of STAT3-ferroptosis regulatory axis alleviated chemoresistance and ferroptosis activity affected oxaliplatin resistance [23, 24]. Since ferroptosis exerts synergistic effects in combination with currently approved treatment [25], which gives us a hint for its potential application in clinic.

Here, this study demonstrates that BAP31 expression increased in GC and closely related with worse prognosis. BAP31 overexpression accelerated GC cell growth and inhibited ferroptosis, whereas BAP31 knockdown suppressed cell growth and promoted ferroptosis. Mechanistically, BAP31 was transcriptionally activated by HNF4A, and further inhibited ferroptosis and facilitated cell proliferation through binding with voltage-dependent anion channel 1 (VDAC1) by promoting its degradation through the ubiquitin-proteasome system and affecting VDAC1 oligomerization. Overall, we depicted the mechanism of BAP31 in the development of GC and tested the feasibility of treating GC with ferroptosis inducer in combination with chemotherapy.

It has been reported that BAP31 is increased in multiple cancers [10], including colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), brain lower grade glioma (LGG), etc., which is corroborated by data of different cohorts from TCGA (The Cancer Genome Atlas) database (Supplementary Fig. S1A). To explore the biological behavior of BAP31 in GC, the expression of BAP31 in GC was analyzed using TCGA. The expression of BAP31 was higher in GC tissues compared with noncancerous tissues (Fig. ​(Fig.1A).1A). Furthermore, the mRNA level of BAP31 positively related to tumor malignancies (Fig. ​(Fig.1B).1B). In addition, the expression of BAP31 was increased in GC tissues compared with noncancerous tissues using {"type":"entrez-geo","attrs":{"text":"GSE66229","term_id":"66229"}}GSE66229 datasets (Fig. ​(Fig.1C).1C). Western blots were conducted in GC samples from our laboratory, showing that BAP31 expression in GC tissues was higher than that in noncancerous tissues (Fig. ​(Fig.1D).1D). Besides, immunohistochemistry (IHC) assay implied a higher BAP31 protein expression in GC tumor tissues at a ratio of 57.6% (53/92) (Fig. ​(Fig.1E,1E, F). Overall, above studies suggest that BAP31 expression is significantly enhanced in GC.

A BAP31 was analyzed in GC tissues and in non-cancerous gastric tissues derived from TCGA data. B TCGA datasets were used to analyze the expression of BAP31 in different GC stages, and nodal metastasis. C BAP31 expression in GC tissues as well as noncancerous gastric tissues was analyzed using the {"type":"entrez-geo","attrs":{"text":"GSE66229","term_id":"66229"}}GSE66229 dataset. D An analysis of BAP31 expression in GC tissues compared to noncancerous gastric tissues by western blot. E Representative IHC images showing the level of BAP31 in GC tissues and the corresponding noncancerous tissues. F Pie chart illustrating the proportion of upregulation, unchanged and downregulation in BAP31 for comparison between GC tissues and noncancerous tissues. G Kaplan–Meier analysis of overall survival in GC patients with differential BAP31 expression in 159 samples. Univariate (H) and multivariate (I) Cox proportional hazard analyses were conducted to evaluate the HR of BAP31 in GC. *p