Cetuximab and Paclitaxel Drug Response in Head and Neck Tumor Stem Cells

Abstract

Head and neck cancer (HNC) is one of the most common types of cancer in the world, characterized by resistance to conventional therapies and an unfavorable prognosis due to the presence of tumor stem cells (TSCs). TSCs are cell subpopulations with high potential for invasion, migration, and metastasis, being responsible for the initiation and dissemination of cancer. This study aimed to evaluate the efficacy of treatments with cetuximab and paclitaxel, alone and in combination, in TSCs from oral cavity (SCC-28) and hypopharynx (FADU) cancer cell lines. In addition, the influence of the gene and protein expression of EGFR, NTRK2 (TRKB), KRAS, and HIF-1α on the response to treatments was investigated. TSCs were identified based on ALDH staining, and cell viability assays (MTS) indicated that both TSCs and non-TSCs showed resistance to cetuximab monotherapy, while paclitaxel, either alone or in combination with cetuximab, was more effective in reducing cell viability. Real-time PCR and Western blot analysis revealed increased expression of KRAS and HIF-1α in TSCs, suggesting their possible association with treatment resistance. The results of this study point to specific molecular factors that influence therapeutic responses in HNC, with an emphasis on the efficacy of drug combinations to overcome TSC resistance. The identification of these molecular mechanisms may provide guidelines for the development of more targeted and effective therapies against HNC, improving clinical management and patient prognoses.

Keywords: cancer treatment; cell signaling pathways; chemotherapy; gene expression; head and neck cancer; tumor stem cells.

Figure 5

Signaling pathway activated by EGFR and TRKB receptors resulting in cell proliferation. (A) TK receptors are activated by endogenous binding, initiating a cascade downstream of cell proliferation. After binding to the growth factor, TK receptors are phosphorylated and activate KRAS, which can activate HIF-1A via the MAPK or PI3K pathways. HIF-1A induces gene transcription leading to cell proliferation, cell survival, drug resistance, and angiogenesis. Adapted by Amatu et al., 2016 [25,43]. (B) EGFR and TRKB crosstalk; cetuximab drug binding and EGFR receptor inhibition, blocking EGFR-dependent downstream cascade. TRKB activation, initiating cell proliferation processes, even when cetuximab treatment inhibits EGFR. Adapted by Gotz and Sentner, 2014 [23]. (C) Process of cell proliferation and targeting of drugs that act on cellular microtubules, blocking the cellular division process in the G2–M phase in the anaphase stage, causing cell death. Adapted by Souza, MVN 2004 [41]. (D) Polymerization and depolymerization process of alpha and beta tubulin heterodimers, forming cell microtubules, which are crucial for cell division; paclitaxel binding in alpha–beta tubulin heterodimers, leading to microtubule stabilization and causing cell death. Adapted by Souza, MVN 2004 [41] and Sueth-Santiago, V et al., 2017 [44].

Figure 1

Confirmation of stemness properties in SCC-28 and FADU cells. The scales of pictures were captured using an optical microscope (×100), and the mean of cells in each group (TSCs and non-TSCs) was compared via a statistical analysis (A) Cell invasion assay. (B) Cell migration assay. (C) Tumor-sphere-formation assay. Cells were photographed at 0 h (SCC-28: (a,c); FADU: (e,g)) and after 120 h (SCC-28: (b,d); FADU: (f,h)) of cultivation. The mean of cells in each group (TSCs and non-TSCs) at 120 h was compared through a statistical analysis. Statistically significant differences were determined using the t-test. Note: * p < 0.050; ** p < 0.010; **** p < 0.0001.

Figure 2

Cell viability after 24 h of treatment in both SCC-28 and FADU TSCs and non-TSCs after drug therapy. (A) Responses to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel in the TSC subpopulation of the SCC-28 cell line compared with untreated cells (B) Responses to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel in the non-TSC subpopulation of the SCC-28 cell line compared with untreated cells. (C) Comparison of response to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel between TSCs and non-TSCs of the SCC-28 cell line. (D) Responses to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel in the TSC subpopulation of the FADU cell line compared with untreated cells. (E) Responses to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel in the non-TSC subpopulation of the FADU cell line compared with untreated cells. (F) Comparison of response to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel between TSCs and non-TSCs of the FADU cell line. Note: ** p < 0.010; *** p < 0.001; **** p < 0.0001; ns: no significance.

Figure 3

Comparison between treatments in each TSC and non-TSC population, as well as comparison of the therapeutic responses between both SCC-28 and FADU cell lines. (A) Comparison of the response of TSC and non-TSC subpopulations of the SCC-28 cell line to treatments with cetuximab, paclitaxel, and cetuximab plus paclitaxel. (B) Comparison of the response of TSC and non-TSC subpopulations of the FADU cell line to treatments with cetuximab, paclitaxel, and cetuximab plus paclitaxel. (C) Comparison of the response to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel between TSC subpopulations of SCC-28 and FADU lines. (D) Comparison of response to treatment with cetuximab, paclitaxel, and cetuximab plus paclitaxel between non-TSC subpopulations of SCC-28 and FADU lines. Note: ** p < 0.010; **** p < 0.0001; ns: no significance.

Figure 4

Relative gene expression levels and protein expression in SCC-28 and FADU cells. Data are presented as the RQ median and the comparison between TSCs and non-TSCs (median = 1). (A) SCC-28 gene expression of EGFR, KRAS, and HIF-1α. The NTRK2 gene presented late expression in both TSCs and non-TSCs. (B) FADU gene expression of EGFR, NTRK2, KRAS, and HIF-1α. (C) SCC-28 Western blot analysis of EGFR, TrkB, KRAS, HIF-1α, and β-actin expression, and histogram showing the comparison between the protein band intensity for both TSCs and non-TSCs. (D) FADU Western blot analysis of EGFR, TrkB, KRAS, HIF-1α, and β-actin expression, and histogram showing the comparison between the protein band intensity for both SCC-28 TSCs and non-TSCs. The band intensity for TSCs and non-TSCs was measured using ImageJ and normalized to β-actin. Original figures can be found in Supplementary Materials File S2. Note: * p < 0.050; ** p < 0.010; *** p < 0.001; ns: no significance.