TCRP1 transcriptionally regulated by c-Myc confers cancer chemoresistance in tongue and lung cancer

Abstract

Previously, we cloned a new gene termed 'tongue cancer resistance-associated protein 1' (TCRP1), which modulates tumorigenesis, enhances cisplatin (cDDP) resistance in cancers, and may be a potential target for reversing drug resistance. However, the mechanisms for regulating TCRP1 expression remain unclear. Herein, we combined bioinformatics analysis with luciferase reporter assay and ChIP assay to determine that c-Myc could directly bind to TCRP1 promoter to upregulate its expression. TCRP1 upregulation in multidrug resistant tongue cancer cells (Tca8113/PYM) and cisplatin-resistant A549 lung cancer cells (A549/DDP) was accompanied by c-Myc upregulation, compared to respective parental cells. In tongue and lung cancer cells, siRNA-mediated knockdown of c-Myc led to decrease TCRP1 expression, whereas overexpression c-Myc did the opposite. Moreover, TCRP1 knockdown attenuated chemoresistance resulting from c-Myc overexpression, but TCRP1 overexpression impaired the effect of c-Myc knockdown on chemosensitivity. Additionally, in both human tongue and lung cancer tissues, c-Myc protein expression positively correlated with TCRP1 protein expression and these protein levels were associated with worse prognosis for patients. Combined, these findings suggest that c-Myc could transcriptionally regulate TCRP1 in cell lines and clinical samples and identified the c-Myc-TCRP1 axis as a negative biomarker of prognosis in tongue and lung cancers.

Figure 1

TCRP1 is upregulated by c-Myc in tongue and lung cancer cells. (A) A sketch map of potential TCRP1 promoter predicted by several online program. The transcriptional start site (TSS) of TCRP1 promoter was defined as‘+1’. (B) A schematic representation of c-Myc binding site in putative TCRP1 promoter. C-Myc also is known as Myc. (C) ChIP assay was performed in tongue cancer cell Tca8113/PYM. Pol IIcontacts to polymerase II. (D) TCRP1 and c-Myc expression were measured by real-time RT-PCR and western blot assay in tongue and lung cancer cells. **P < 0.01. (E,F) Plasmid pMxs-h-c-Myc and control pMxs-h were transfected into tongue cancer cell Tca8113 and lung cancer cell A549, siRNAs targeting c-Myc together with its control were transfected into Tca8113/PYM and A549/DDP cells, 48 h later, the mRNA and protein expression levels of TCRP1 and c-Myc were estimated in these cells. **P < 0.01. (G) A schematic diagram of c-Myc wild type and mutant binding site in TCRP1 promoter. Dual-luciferase reporter gene assay was conducted in HEK-293T, tongue and lung cancer cells with overexpression c-Myc or siRNA targeting c-Myc. **P < 0.01.

Figure 2

The c-Myc-TCRP1 axis inhibites sensitivity of tongue and lung cancer cells to cDDP. (A) Transfected plasmid pLEX-TCRP1 and its control into Tca8113 cells, meanwhile transfected siRNAs targeting TCRP1 and its control into Tca8113/PYM cells, 48 h later, western blot assay was used to measure TCRP1 expression levels in these different treated cells. (B) Overexpressed c-Myc together with silencing TCRP1 in parative cells Tca8113 and A549, (C) Silencing c-Myc together with over-expressed TCRP1 in chemoresistance cells Tca8113/PYM and A549/DDP, all these cells and each control group were treated with cDDP for 72 h, and the survival rate of these cells were detected by MTS assay, western blot assay was used to measure Akt activation. **P < 0.01.

Figure 3

c-Myc positively related with TCRP1 and poor clinical outcome of patients. (A) Immunohistochemical assay was used to obtain c-Myc, TCRP1 and cleaved-caspase 3 expression in tongue and lung cancer tissues. Representative images of each protein expression was showed, and the relative expression pattern between TCRP1 and c-Myc or cleaved-caspase 3 were also analyzed. In tongue cancer tissues (B) and lung cancer tissues (C), Kaplan–Meier analysis was performed to estimate OS according to the protein level of TCRP1, c-Myc, even both them.