PLAC8 overexpression correlates with PD-L1 upregulation and acquired resistance to chemotherapies in gallbladder carcinoma

https://doi.org/10.1016/j.bbrc.2019.06.121Get rights and content

Highlights

  • PLAC8 upregulation confers acquired resistance to GEM and OXA in GBC cells.

  • PLAC8 downregulation increases sensitivity to GEM and OXA in GBC cells.

  • PLAC8 overexpression observed in GBC patients positively correlates with PD-L1.

  • PLAC8 confers acquired resistance to GEM and OXA via upregulating PD-L1 expression.

Abstract

Gallbladder carcinoma (GBC) is always diagnosed at an advanced stage, and patients often miss the opportunity for surgery. Gemcitabine (GEM) and platinum-based drugs, including oxaliplatin (OXA), are mainstays of chemotherapy. However, drug resistance causes treatment failure. Hence, salvage mechanisms are critical to improve outcomes. This study revealed the positive correlation between placenta-specific protein 8 (PLAC8) overexpression and PD-L1 overexpression in GBC. Given the roles of PLAC8 and PD-L1 in chemotherapy resistance, GEM-resistant and OXA-resistant cell lines (SGC966GR and SGC966OR, respectively) were established to test whether and how PLAC8 and PD-L1 function in chemotherapy resistance. Drug-insensitive SGC966GR and SGC966OR cells upregulated MRP and MDR1 and had high expression of PLAC8. PLAC8 blockade using siRNA reversed chemotherapy resistance and downregulated MRP and MDR1 in SGC966GR and SGC966OR cells, suggesting that PLAC8 mediates chemotherapy resistance in GBC. Consistent with the increased mRNA levels of PD-L1 after the acquisition of resistance, PLAC8 knockdown reduced PD-L1 mRNA expression in SGC966GR and SGC966OR cells. In conclusion, PLAC8 overexpression in GBC patients positively correlated with PD-L1 expression. PLAC8 conferred resistance to GEM and OXA by upregulating PD-L1 expression, and PLAC8 or PD-L1 blockade may have potential for overcoming chemotherapy resistance, providing therapeutic options for chemotherapy-refractory GBC patients.

Introduction

Gallbladder carcinoma (GBC) patients with atypical symptoms at an early stage are usually diagnosed at a locally advanced or metastatic stage, where conventional chemotherapies remain a mainstay [[1], [2], [3]]. However, limited responses to chemotherapies and a lack of effective targeted therapies result in unsatisfactory survival outcomes in advanced GBC [4,5]. Thus, the mechanisms underlying chemotherapy resistance and strategies for chemotherapy resensitization together with novel potential therapeutic targets urgently need to be studied in GBC.

GEM plus a platinum-based drug serves as the most prevalent chemotherapy scheme in the treatment of advanced GBC in both inoperative patients and those with a high risk of postoperative recurrence [1]. As reported, acquired resistance to GEM and platinum-based drugs in GBC involves disordered drug efflux systems marked by abnormal upregulations of ABC family genes [6], as witnessed in other cancers [7,8]. Nevertheless, therapeutic options for overcoming chemotherapy resistance are absent for these molecular indications. More strategies, especially those associated with specific and druggable targets, against chemotherapy-resistant GBC need to be developed.

Developing novel therapeutic strategies against GBC is also necessary for improving clinical outcomes. Recently, immune checkpoint blockade in the form of PD-1/PD-L1 inhibitors has become the most promising therapeutic option for GBC [9,10]. Of interest, PD-1/PD-L1 blockade has a tight link with chemotherapy efficacy. The positive responses to salvage chemotherapy following exposure to PD-1/PD-L1 inhibitors in patients with NSCLC [11] suggest that sequential schemes involving PD-1/PD-L1 inhibitors followed by chemotherapy can beneficially result in immunotherapy-activated chemotherapy sensitivity in cancers. Additionally, acquired chemotherapy resistance can be initiated by PD-L1, and the combination of PD-1/PD-L1 inhibitors with chemotherapy is a promising therapeutic option for treating chemotherapy-resistant NSCLC [12,13]. However, the role of PD-L1 in chemotherapy resistance in GBC remains unclear. In addition, upstream regulators of PD-L1 expression are often considered better candidates for PD-1/PD-L1 blockade-containing combination strategies in cancers [14,15]. Thus, identifying novel molecules upstream of PD-L1 is of significance in GBC. Apart from PD-1/PD-L1 inhibitors, there are almost no novel therapeutic targets found in GBC. Thus, novel molecular targets, especially those related to PD-L1 levels and chemotherapy resistance, deserve and in-depth analysis in GBC.

In this work, we identified a novel potential molecular biomarker, placenta-specific protein 8 (PLAC8), that is highly expressed in GBC using TMAs of surgical specimens. We then examined the relationship of PLAC8 levels with PD-L1 expression in GBC tissues as well as their roles in chemotherapy resistance using our established GEM-resistant and OXA-resistant GBC preclinical models. Our work sheds light on the therapeutic potential of targeting PLAC8 and PD-L1 in GBC, especially in chemotherapy-resistant patients.

Section snippets

Cell culture

The human gallbladder carcinoma cell line SGC996 was purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1x penicillin-streptomycin (HyClone) at 37 °C in a humidified 5% CO2 atmosphere.

Establishment of drug-resistant cells

For up to 2 months, SGC996 cells were treated with GEM at concentrations ranging from 5 μM to 40 μM or OXA at concentrations ranging from 2 μM to 20 μM; the resulting drug-resistant cells were named SGC996GR

PLAC8 protein expression was higher in tumor tissues than in adjacent normal tissues and positively correlated with PD-L1 expression in GBC

PLAC8, an oncogene, is universally overexpressed among various cancer types and can serve as a prognostic biomarker to predict poor prognosis [[17], [18], [19], [20], [21]]. However, the expression of PLAC8 in GBC remains uninvestigated. Thus, PLCA8 expression was evaluated in 20 pairs of GBC tissues and normal tissues arranged in TMAs. Similar to published results [22], PLAC8 was primarily expressed in the cytosol in GBC tissues (Fig. 1a). Of the evaluable samples, one GBC sample (5%) showed

Discussion

The majority of patients with GBC are diagnosed at an advanced stage, with a 5-year survival rate of approximately 5%, and limited therapeutic options are available for them [2,3]. Chemotherapies, typically GEM and platinum-based drugs, are common therapy options and benefit patients to some extent, but chemotherapy resistance is frequently a challenge [1,5]. Thus, novel therapeutic targets and relevant treatment strategies that target the mechanisms underlying chemotherapy resistance should be

Data availability

The assays used to support the findings of this study are included within the article.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by Zhongshan Hospital, Biliary Tract Diseases Institute, Fudan University, Shanghai 200032, P. R. China.

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