The most important member of the receptor tyrosine kinases (RTK) family is the epidermal growth factor receptor (EGFR). Cetuximab, a chimeric IgG1 monoclonal antibody against EGFR, was first approved by the U.S. Food and Drug Administration (FDA) in 2004 for patients with irinotecan-resistant colorectal cancer [26], and two years later was authorized for the treatment of locally advanced HNSCC [27]. Even 90% of HNSCC patients have overexpression of the EGFR [28]. Unfortunately, cetuximab has only shown a 20% positive response rate in patients with HPV-negative tumors and only a marginal improvement in combination with radiotherapy and platinum-based chemotherapy [29,30]. Panitumumab is another monoclonal antibody against EGFR [31].

Irreversible EGFR inhibitors act on the intracellular domain, inhibiting the cytoplasmic tyrosine kinase domain. Erlotinib is the most respected representative, and was, for instance, combined with standard docetaxel/cisplatin chemotherapy for recurrent/metastatic (R/M) HNSCC [32]. The combined treatment achieved a response rate of 62% (8% complete response and 54% partial response), which is greater than the previous trial’s response rate of 40% for docetaxel/cisplatin chemotherapy.

Many studies have found that HNSCC and other EGFR-dependent tumors are resistant to EGFR-inhibitory therapy. Yamaoka et al. (2017) summarized four general mechanisms of anti-EGFR antibody and EGFR tyrosine kinase inhibitor resistance in cancer cells: (i) secondary mutations in the EGFR gene; (ii) resistance to apoptotic cell death; (iii) phenotypic transformation (e.g., tumor cells activating stem cell-like characteristics); and finally, (iv) activation of alternative signaling pathways [33].

The active state of EGFR triggers a cascade of intracellular responses. As a result, in many cases of molecular abnormalities, even effective EGFR attenuation cannot influence the downstream activation of altered signal transduction elements. Therefore, there is a recurrence of a tumor that is resistant to previously used therapeutic procedures.

RAS is a key player in the EGFR signal transduction. The HRAS mutations are called “undruggable”, but advances in the high-resolution understanding of RAS isoform structure provide hope for developing personalized therapies for patients with RAS-dependent cancers [34]. Fortunately, post-transcriptional farnesylation is required for RAS protein to be anchored to the inner side of the cell membrane, which is crucial for EGFR signal transduction. A phase II clinical trial of tipifarnib (inhibitor of farnesyltransferase) involving 30 patients with R/M HNSCC revealed positive response in patients with HRAS mutations [35].

EGFR-dependent RAS activation stimulates two critical intracellular signaling pathways, RAS/RAF/MAPK and PI3K/Akt/mTOR [36].

In brief, this pathway creates kinase cascades and finally activates extracellular signal-regulated kinases (ERK) [37], which translocate from the cytoplasm to the nucleus to induce specific genes expression [38]. In HNSCC, attempts were made to target this kinase cascade by inhibiting the RAF and MEK proteins. For instance, Sorafenib—a RAF kinase, vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) inhibitor [39]—was evaluated in phase II clinical trial of patients with R/M HNSCC and resulted in a partial response or disease stabilization in 40.7–51% of patients [40,41].

Phosphoinositide 3-kinase (PI3K) class IA comprises the p110α/β/δ catalytic subunit and the p85 regulatory subunit. Phosphatidylinositol 3,4,5-trisphosphate, converted from phosphatidylinositol 4,5-bisphosphate, activates downstream signaling factors such as Akt. Another kinase, the mammalian target of rapamycin (mTOR), is the main effector of Akt kinase [42]. Mutations in the PI3K catalytic subunit p110α are the most common genetic abnormality observed in HNSCC. Alpelisib (NVP-BYL719) is the first FDA-approved p110α inhibitor for the treatment of hormone receptor-positive, HER2-negative, PI3K catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer, and it may be useful in HNSCC as well [43,44].

Akt phosphorylates a variety of targets, including tuberous sclerosis complex 2 (TCS2), which, along with TCS1, inhibits the activity of the mTOR complex (mTORC) [45]. Based on the U.S. National Library of Medicine online (https://clinicaltrials.gov) database of clinical studies, Akt inhibitors-ipatasertib (GDC-0068) and capivasertib (AZD5363), are tested for R/M HNSCC in mono-treatment (NCT02465060 and NCT02465060, respectively), followed by ipatasertib in combination with cisplatin and radiotherapy (NCT05172245).

Finally, mTOR inhibition may be used to target this pathway, e.g., by everolimus. A meta-analysis of studies involving mTOR inhibition confirms that monotherapy cannot improve the prognosis of HNSCC patients but can accelerate partial tumor response when combined with other anticancer agents [46].

Other RTK, in addition to EGFR, may be promising pharmacological targets in HNSCC. In some tumors, the fibroblast growth factor receptor (FGFR) is overexpressed and partially amplified [47] and was linked to poor overall survival and disease-free survival in HPV-negative patients. The small molecule, AZD4547, is a potential FGFR inhibitor, which was found to decrease the growth of HNSCC cells in vitro [48].

The VEGFR signaling orchestrates neovascularization of growing tumors [49]. Because RAS, PI3K, and STAT3 proteins are downstream effectors of VEGFR [50], its simulation promotes many other tumor-promoting features controlled by those pathways. Bevacizumab, a humanized monoclonal antibody against VEGF, is frequently examined in clinical trials; for instance, it was combined with EGF-receptor-targeted therapy based on cetuximab [51] or erlotinib [52], which benefits patients.

The PDGFR signaling, among others, influences Akt-dependent activation of pro-oxidative NF-κB signaling [53]. Overexpression of PDGF and its receptor has been associated with neck lymph node metastasis, advanced TNM stage, and poor survival in HNSCC patients [54]. Multifunctional kinase inhibitors are currently being used to target this receptor along with other RTKs. Imatinib, a PDGF(R) and VEGF(R) inhibitor suppressed their expression synergistically in vitro [55].

In HNSCC, the hepatocyte growth factor/mesenchymal-epithelial-transition factor (HGF/c-MET) pathway promotes PI3K/Akt, RAS/MAPK, STAT3, and Src/NF-κB intracellular signaling, resulting in cancer cell proliferation and apoptosis avoidance, followed by extensive growth and metastasis [56,57]. Wang et al. (2021) used three c-Met inhibitors (crizotinib, tivantinib, and cabozantinib) in combination with the pan-HER inhibitor afatinib. In HNSCC cell lines, xenografts, and patient-derived xenograft animal models, the drugs’ combination exceeds monotherapy regarding anticancer efficacy, confirming the significance of further clinical trials [58].

STAT canonical signaling can be activated by RTK, as described in this section, resulting in neovascularization, increased cell proliferation, survival, and even immune response evasion [59]. The nuclear accumulation of phosphorylated STAT3 has been identified as a prognostic marker in the early premalignant stages of HNSCC [60]. STAT5 inhibitor 573108, in combination with radiotherapy, was found to improve cell survival in a panel of HNSCC cell lines [61].

Cancer stem cells (CSC) are a subpopulation of cells that express specific extracellular and molecular markers and can self-renew [62,63,64]. After temporary tumor bulk reduction, conventional anticancer therapy that does not affect CSC leads to tumor recurrence with an enriched, therapy-resistant CSC population.

The NOTCH pathway regulates body pattern formation, cell fate, and proliferation during embryogenesis, and stem cell activity in both early and adult organisms [65]. The global mutation rate of NOTCH1 is approximately 15%, making this gene one of the most frequently mutated in HNSCC [66]. The NOTCH1 gene was thought to be a tumor suppressor due to the high percentage of mutations in HNSCC [67], but this pathway can be induced in tumors as well [68]. The NOTCH pathway promotes the self-renewal capacity of HNSCC cells, as evidenced by increased expression of Oct4, Sox2, and CD44 stemness markers [69].

The Wnt/β-catenin signaling is essential for cell differentiation and proliferation during embryogenesis and in proliferative tissues in adulthood, including the stem cell subpopulation [70,71]. This signaling is extensively activated in colorectal cancers, but its dysregulation at various levels of signal transduction is also critical for the development of HNSCC [72]. In particular, the porcupine inhibitor (IWP-2) and the inhibitor of the interaction between β-catenin and the CREB binding protein (PRI-724) effectively inhibited HNSCC cell lines [73].

The Hedgehog (Hh) canonical pathway is activated by the Sonic Hedgehog (SHh) ligand and is present in various tissues/organs during development and in the adult organism [74,75]. The significance of the Hh pathway in the development of basal cell carcinoma of the head and neck was practically confirmed by the FDA’s approval of the Hh signaling inhibitor, vismodegib, in 2012 [76]. Several studies [77,78,79,80] have identified active Hh signaling as a negative prognostic marker for HNSCC patients and multi-drug resistance. Furthermore, in HNSCC, Hh signaling is strongly linked to CSC markers [81].

NOTCH signaling activation can upregulate components of the Wnt and Hh pathways, and further crosstalk between those signaling pathways supports the maintenance and development of HNSCC by promoting the activity of CSC [82]. In addition, it is possible that also the Hippo pathway, which is involved in organ development, regeneration, and stemness, could be used as a target for HNSCC combinatorial therapy, while its crosstalk with NOTCH, Wnt, and Hh signaling has been demonstrated [83,84]. Finally, because transforming growth factor-β (TGF-β) is a regulatory cytokine involved in the control of CSC and immune cells [85,86], it is a good target for innovative combinatorial HNSCC treatment.

The use of two monoclonal antibodies against programmed cell death 1 (PD-1) was a practical success in overcoming an abnormal immune response of HNSCC cells. Cancer cells produce excessive PD-1 ligands (PD-L1/2), which binds to PD-1 receptors on the surface of T-cells. As a result, T-cell activity, proliferation, cytokine secretion, and overall survival are all affected [87,88]. Pembrolizumab is an FDA-approved IgG4-κ humanized monoclonal antibody against PD-1, activating the immune response [89,90]. Another IgG4 antibody, nivolumab, was also approved to treat HNSCC [91].

Otto Warburg observed specific energy metabolism in cancer cells using glycolysis and fermentation, despite access to oxygen [92]. Nowadays, we have a much better understanding of the so-called Warburg effect. Glycolysis, glutaminolysis, NAD synthesis, tricarboxylic acid cycle, mitochondrial activity, changes in intra- and extracellular pH, lipid and amino acid metabolism, and control of master regulators of energy metabolism such as c-Myc, HIF-1α, Akt, or sirtuins are examples of metabolic targets [93,94,95]. Some commonly used chemotherapeutics target metabolism (e.g., methotrexate - folic acid metabolism) are registered for non-cancer purposes and used in antitumor procedures (e.g., metformin related with glucose metabolism), or are in clinical trials (e.g., AZD-3965 inhibiting lactate transporter MCT1) [96]. The reorganization of cancer cells’ metabolism cooperates with other molecular abnormalities and should be considered an adjuvant therapy in most cases.

Figure 1 represents the targets, enriched with other possible targets of HNSCC therapy [97,98,99,100,101,102,103].