Cancer-associated fibroblasts contribute to cisplatin resistance by modulating ANXA3 in lung cancer cells

Abstract

Cancer tissues consist of cancer cells, surrounding stromal cells and the extracellular matrix. Cancer-associated fibroblasts (CAF) are one of the key components of stromal cells. CAF have a great impact on the behavior of cancer cells, including proliferation, invasion, metastasis and chemoresistance in many ways. However, the underlying mechanism had not been fully elucidated. In this study, we investigated the role of CAF in cisplatin resistance of lung cancer cells. By using conditioned medium from CAF (CAF-CM), we found that CAF decreased the sensitivity of lung cancer cells to cisplatin. RNA sequencing results showed that CAF expressed a higher level of Annexin A3 (ANXA3) than normal fibroblasts (NF), and CAF-CM incubation increased the ANXA3 level in lung cancer cells. Overexpression of ANXA3 in lung cancer cells increased cisplatin resistance and activated c-jun N-terminal kinase (JNK), whereas knockdown of ANXA3 increased cisplatin sensitivity. Further study showed that CAF-CM enhanced cisplatin resistance by inhibiting cisplatin-induced apoptosis, determined by repression of caspase-3 and caspase-8, through activation of the ANXA3/JNK pathway. Conversely, suppression of JNK activation by specific inhibitor retarded the effect of CAF-CM and ANXA3 on cisplatin sensitivity. Taken together, our study demonstrated that CAF potentiated chemoresistance of lung cancer cells through a novel ANXA3/JNK pathway both in vitro and in vivo, suggesting ANXA3 could be a potential therapeutic target for the treatment of chemoresistant cancer.

Keywords: ANXA3; JNK; cancer-associated fibroblast; cisplatin; lung cancer.

Figure 1

Cancer‐associated fibroblasts (CAF) enhanced cisplatin resistance of lung cancer cells. Lung cancer cells were cultures in CAF‐CM and treated with 0‐80 μmol/L cisplatin for 48 h. Cell viability was detected by CCK‐8 assay. Values represent the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 2

Cancer‐associated fibroblasts (CAF) elevated the expression of ANXA3 in lung cancer cells. A,B, Gene expression in CAF and normal fibroblasts (NF) was assessed by RNA‐Seq. The differential gene expression and the ontology analysis were performed. C, The expression of ANXA3 in paired CAF and NF were detected by western blotting. D, The mRNA expression of ANXA3 in paired CAF and NF were detected by quantitative PCR. E, The expression of ANXA3 in lung cancer cell lines was detected by western blotting. F, The expression of ANXA3 in lung cancer cells cultured with CAF‐CM was detected by western blotting

Figure 3

Elevated expression of ANXA3 in lung cancer cells was mediated by ANXA3 secretion in cancer‐associated fibroblasts (CAF). A,B, ANXA3 was overexpressed or knocked down in CAF, the CAF‐CM was collected and incubated with lung cancer cells. The expressions of ANXA3 in lung cancer cells were detected by western blotting. C, Recombinant ANXA3 was added to DMEM and incubated with lung cancer cells. The expressions of ANXA3 in lung cancer cells were detected by western blotting. ANXA3 neutralizing antibody (500 ng/mL) was added to CAF‐CM and incubated with lung cancer cells. The expression of ANXA3 in lung cancer cells was detected by western blotting

Figure 4

ANXA3 mediated the effect of cancer‐associated fibroblasts on cisplatin resistance of lung cancer cells. A, A549 and H661 cells were transfected with pANXA3, and SK‐MES‐1 cells were transfected with ANXA3‐siRNA duplex; ANXA3 expression were analyzed by Western blotting. B,C, After transfection cells were treated with 0‐80 μmol/L cisplatin for 48 h, cell viability was detected by CCK‐8 assay. Values represent the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 5

Cancer‐associated fibroblasts (CAF) enhanced cisplatin resistance of lung cancer cells. Lung cancer cells were cultured in CAF‐CM with ANXA3 neutralizing antibody (500 ng/mL) and treated with 0‐80 μmol/L cisplatin for 48 h. Cell viability was detected by CCK‐8 assay. Values represent the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 6

Cancer‐associated fibroblasts (CAF) activated the JNK/survivin pathway by upregulating ANXA3. A, A549 and H661 cells were incubated with CAF‐CM for 24 h, then treated with 15 μmol/L cisplatin for 36 h. The protein expressions were detected by western blotting. B, A549 and H661 cells were transfected with pANXA3. SK‐MES‐1 cells were transfected with ANXA3‐siRNA duplex, then treated with 15 μmol/L cisplatin for 36 h. The protein expressions were detected by western blotting

Figure 7

JNK/Survivin pathway mediated the effect of cancer‐associated fibroblasts (CAF) on cisplatin resistance of lung cancer cells. A, A549 and H661 cells were cultured in CAF‐CM and pre–treated with 2 μmol/L JNK inhibitor SP600125 for 1 h, then were treated with 0‐40 μmol/L cisplatin for 48 h. Cell viability was detected by CCK‐8 assay. B, A549 and H661 cells were transfected with pANXA3 and pre–treated with 2 μmol/L SP600125 for 24 h, then were treated with 0‐40 μmol/L cisplatin for 48 h. Cell viability was detected by CCK‐8 assay. C, Protein expressions were detected by western blotting. D, Cells were stained with Annexin/PI after treatment and the apoptotic cells were analyzed by flow cytometry. Values represent the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 8

Cancer‐associated fibroblasts (CAF) enhanced cisplatin resistance of lung cancer in vivo. 3 × 10⁶ A549 cells, alone or with 9 × 10⁶ CAF (ratio1:3) were injected subcutaneously into the back of BALB/c nude mice. Mice were treated with PBS or cisplatin (3 mg/kg body weight) for 6 wk. A, At the end of treatment, tumor formation was inspected and tumors were excised. B, Tumor volumes were measured every week. C, The protein expressions in tumor tissues were detected by western blotting

Figure 9

A proposed model of cancer‐associated fibroblasts enhanced cisplatin resistance in lung cancer cells