In methicillin-sensitive Staphylococcus aureus (MSSA), the tricarboxylic acid (TCA) cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG. However, methicillin-resistant S. aureus (MRSA) produce a primarily proteinaceous biofilm matrix, and contribution of the TCA-cycle therein remains unclear. Utilizing USA300-JE2 Tn-mutants (NARSA) in genes encoding TCA- and urea cycle enzymes for transduction into a prolific biofilm-forming USA300 strain (UAS391-Ery^s), we studied the contribution of the TCA- and urea cycle and of proteins, eDNA and PIA/PNAG, to the matrix. Genes targeted in the urea cycle encoded argininosuccinate lyase and arginase (argH::Tn and rocF::Tn), and in the TCA-cycle encoded succinyl-CoA synthetase, succinate dehydrogenase, aconitase, isocitrate dehydrogenase, fumarate hydratase class II, and citrate synthase II (sucC::Tn, sdhA/B::Tn, acnA::Tn, icd::Tn, fumC::Tn and gltA::Tn). Biofilm formation was significantly decreased under no flow and flow conditions by argH::Tn, fumC::Tn, and sdhA/B::Tn (range OD₄₉₂ 0.374−0.667; integrated densities 2.065−4.875) compared to UAS391-Ery^S (OD₄₉₂ 0.814; integrated density 10.676) (p ≤ 0.008). Cellular and matrix stains, enzymatic treatment (Proteinase K, DNase I), and reverse-transcriptase PCR-based gene-expression analysis of fibronectin-binding proteins (fnbA/B) and the staphylococcal accessory regulator (sarA) on pre-formed UAS391-Ery^s and Tn-mutant biofilms showed: (i) < 1% PIA/PNAG in the proteinaceous/eDNA matrix; (ii) increased proteins under no flow and flow in the matrix of Tn mutant biofilms (on average 50 and 51 (±11)%) compared to UAS391-Ery^s (on average 22 and 25 (±4)%) (p < 0.001); and (iii) down- and up-regulation of fnbA/B and sarA, respectively, in Tn-mutants compared to UAS391-Ery^S (0.62-, 0.57-, and 2.23-fold on average). In conclusion, we show that the biofilm matrix of MRSA-USA300 and the corresponding Tn mutants is PIA/PNAG-independent and are mainly composed of proteins and eDNA. The primary impact of TCA-cycle inactivation was on the protein component of the biofilm matrix of MRSA-USA300.

The epidermal growth factor receptor (EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR-targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti-EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first-generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab-sensitive, intrinsically cetuximab-resistant, and acquired cetuximab-resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab-sensitive, intrinsically cetuximab-resistant, and acquired cetuximab-resistant HNSCC cell lines, independent of the HPV status. However, cross-resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G₀ /G₁ cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance.

Increased expression of the epidermal growth factor receptor (EGFR) is observed in more than 90% of all head and neck squamous cell carcinomas (HNSCC). Therefore, EGFR has emerged as a promising therapeutic target. Nevertheless, drug resistance remains a major challenge and an important potential mechanism of drug resistance involves the hypoxic tumor microenvironment. Therefore, we investigated the cytotoxic effect of the EGFR-targeting agents cetuximab and erlotinib under normoxia versus hypoxia. Three cetuximab-sensitive HNSCC cell lines (SC263, LICR-HN2 and LICR-HN5) were treated with either cetuximab or erlotinib. Cells were incubated under normal or reduced oxygen conditions (<0.1% O2) for 24 or 72 h immediately after drug addition. Cell survival was assessed with the sulforhodamine B assay. Cetuximab and erlotinib established a dose-dependent growth inhibition under both normal and prolonged reduced oxygen conditions in all three HNSCC cell lines. However, a significantly increased sensitivity to cetuximab was observed in SC263 cells exposed to hypoxia for 72 h (p = 0.05), with IC50 values of 2.38 ± 0.59 nM, 0.64 ± 0.38 nM, and 0.10 ± 0.05 nM under normoxia, hypoxia for 24 h and hypoxia for 72 h, respectively. LICR-HN5 cells showed an increased sensitivity towards erlotinib when cells were incubated under hypoxia for 24 h (p = 0.05). Our results suggest that both EGFR-inhibitors cetuximab and erlotinib maintain their growth inhibitory effect under hypoxia. These results suggest that resistance to anti-EGFR therapy in HNSCC is probably not the result of hypoxic regions within the tumor and other mechanisms are involved.

Polo-like kinase 1 (Plk1), a master regulator of mitotic cell division, is highly expressed in non-small cell lung cancer (NSCLC) making it an interesting drug target. We examined the in vitro therapeutic effects of volasertib, a Plk1 inhibitor, in combination with irradiation in a panel of NSCLC cell lines with different p53 backgrounds. Pretreatment with volasertib efficiently sensitized p53 wild type cells to irradiation. Flow cytometric analysis revealed that significantly more cells were arrested in the G₂/M phase of the cell cycle after the combination therapy compared to either treatment alone (p < 0.005). No significant synergistic induction of apoptotic cell death was observed, but, importantly, significantly more senescent cells were detected when cells were pretreated with volasertib before irradiation compared to both monotherapies alone (p < 0.001), especially in cells with functional p53. Consequently, while most cells with functional p53 showed permanent growth arrest, more p53 knockdown/mutant cells could re-enter the cell cycle, resulting in colony formation and cell survival. Our findings assign functional p53 as a determining factor for the observed radiosensitizing effect of volasertib in combination with radiotherapy for the treatment of NSCLC.