In this study, we used NSCLC cancerous tissues and the A549 cell line to elucidate the implications of TNF-α during the inducement of the NF-κB/PXR pathway and their correlations with chemotherapy resistance. We found that TNF-ICs was negatively associated with PXR and chemosensitivity. Moreover, we discovered that TNF-α secreted by inflammatory stromal cells had a higher effect in controlling chemotherapy resistance compared to TNF-α secreted by tumor cells. TNF-α secreted by inflammatory stromal cells predominantly affects chemotherapy resistance via regulating NF-κB/PXR pathway and PXR transcripts, which was further confirmed in vitro. TNF-α treatment on the A549 cell line triggered the upregulation and activation of NF-κB and reduced the expression levels of PXR as well as its target gene ABCB1. Our results showed that within the solid tumor of NSCLC, exogenous TNF-α secreted by stroma might lead to repressive effects on the chemotherapy resistance phenotype of NSCLC cells. Moreover, NF-κB/PXR pathway activation resulted in a key modulation mechanism involved.
Resistance to therapeutic agents is a major factor leading to an unfavorable outcome in anti-cancer therapy. To date, accumulating evidence has suggested the importance of TNF-α in inducing drug resistance. TNF-α and MMP-9 baseline levels were found to be significantly elevated in metastatic renal-cell carcinoma resistant to sunitinib[17]. The combined application of anti-TNF-α drugs demonstrated a promising therapeutic direction for improving the efficiency of chemotherapy agents, TKI, and immunotherapy. The combination of anti-TNF-α treatment and chemotherapy can suppress colon cancer cells' survival and reduce drug resistance[8]. The application of anti-TNF-α drugs can also overcome resistance to anti-PD-1 in experimental melanoma in vivo and in vitro[9, 10, 18]. In NSCLC, TNF-α blockade has been identified to enhance EGFR inhibition effectiveness[13].
As a pro-inflammatory cytokine with biphasic functions that modulate both pro-carcinoma and anti-carcinoma downstream, anti-TNF-α therapy has potential risks. It may lead to cellular proliferation and promote malignant transformation. Anti-TNF-α can account for a mild risk of incident cancer in a population of IBD patients with recent malignancy[19]. Moreover, a previous study found that rheumatoid arthritis (RA) patients treated with anti-TNF-α have an increased risk of developing non-melanoma skin cancer, especially squamous cell carcinoma[20]. Therefore, further exploration of the regulating mechanisms of TNF-α is important to enhance drug efficiency and avoid side effects.
Recent results suggested the implications of the NF-κB/PXR signaling pathway involved in TNF-α inducing drug resistance. TNF-α affects drug resistance by modulating the NF-κB/PXR signaling pathway to control the PXR target genes' transcripts, including ABCB1, CYP3A11, ABCG2, and GSTa2[16, 17, 21]. However, the regulating mechanisms of TNF-α in the NF-κB/PXR signaling pathway lack homogeneity. DEN-induced hepatic cancer in mice resulted in increased expression of TNF-α and NF-κB; the expression of PXR was concurrently reduced when compared to control mice[16]. Long-term (24–96 h) TNF-α treatment disrupts NF-κB/p65 activation, reduces the nuclear accumulation of NF-κB/p65, and decreases ABCB1 expression, leading to sensitization towards drug treatment in colon cancer[22]. TNF-α and IL-1β treatment for 72 h induces upregulation of ABCG2 and PXR expression consistent with NF-κB activity in some breast cancers, including MCF7, BT-474, CAL51, 184A1, and HBL100 cells[17]. Our study found that TNF-α treatment causes activation of NF-κB and decreases transcripts and functions of PXR in NSCLC.
In most cancers, TNF-α binds to TNFR1 on the cellular membrane and activates NF-κB signaling in a canonical pathway, thus resulting in transcriptional activities of multiple genes[14, 23, 24]. It has been proved that NF-κB affects gene transcripts of PXR by interrupting the PXR-RXRα interaction. PXR is a member of orphan NRs that shares common structural domains consisting of an N-terminal activation function domain 1 (AF-1), a conserved zinc-finger-type DNA binding domain (DBD), and a C-terminal ligand-binding domain (LBD)[16]. PXR is differentially expressed in human cancers: it is increased in prostate, breast, and endometrial, and decreased in colorectal and cervical cancer[25, 26]. In addition, to regulate genes involved in cellular proliferation, tumor metastasis, and apoptosis, PXR targets multidrug resistance protein 1 (MDR1, also known as ABCB1), a drug efflux transporter that encodes P-GP and serves as a major regulator of drug resistance through pumping out anti-cancer agents. Overexpression of ABCB1 is associated with docetaxel and cisplatin-induced drug resistance, which is a frequent problem in the procedure of chemotherapy in NSCLC. Both docetaxel and cisplatin promote elevated expression of ABCB1 in 3D-cultured NSCLC cells, albeit the latter is not a substrate of ABCB1[27]. Thus, the decreased PXR and ABCB1 expressions in response to TNF-α application indicates repressive roles of TNF-α in chemotherapy resistance which is consistent to the evidence deduced in histologic tissues.
The inflammatory regulator network is a bilateral and complicated process. PXR can mutually interplay with and TNF-α and NF-κB. PXR diminishes the inflammatory injury generally through the negative modulation of NF-κB and TNF-α[26]. Thus, rather than a unilateral observation, research on feedback loops is recommended to identify and provide complete information for anti-cancer treatment. The approach for a clinical application requires more detailed research.
In this study, we used an A549 cell line for detecting inducing roles of TNF-α in vitro. A549 cell line has maximal mRNA and protein expression of PXR among various NSCLC cell lines, including A549, NCI-H358, HCC827, NCI-H1650, and NCI-H1299. Our results demonstrated that despite resembling activation of NF-κB, A549 cells displayed repressed PXR expressions in response to TNF-α inducement compared to activated PXR transcripts in normal controls. The contrary alterations of PXR expression in NSCLC and normal cells suggest TNF-α has differential regulatory roles on the NF-κB/PXR pathway between benign and malignancies; TNF-α treatment for drug resistance may accelerate and deteriorate malignant transformation in normal cells; however, this needs to be further explored. Furthermore, it has been known that cytokines can influence cellular proliferation, drug resistance, as well as apoptosis in both autocrine way and/or paracrine way [17]. Although our results demonstrated that TNF-ICs but not TNF-TCs had negative associations with PXR, which suggests the paracrine way is more likely to involve drug resistance, further experiments are required to determine the roles of autocrine TNF-α in mediating drug resistance of NSCLC.
In conclusion, our data revealed that TNF-α expression in tumor-infiltrating inflammatory cells is a predictive biomarker for chemotherapy drug resistance in NSCLC. NF-κB activation and PXR repression triggered by TNF-α are involved in the underlying molecular mechanism. Our findings proposed that the application of TNF-α inhibitors in anti-cancer therapy may block TNF-α secretion of ICs and improve drug resistance in NSCLC. On the other side, TNF-α treatment may sensitize chemotherapy; yet, adverse effects of cellular proliferation promotion should be taken into consideration.
Our findings further the understanding of the roles of inflammatory cells infiltrating tumors and suggest that more subtle regulation mechanisms of TNF-α should be taken into consideration in the clinical usage of TNF-α or its inhibitors.