Cyclin-dependent kinases (CDK) regulate cell cycle progression. During tumor development, altered expression and availability of CDKs strongly contribute to impaired cell proliferation, a hallmark of cancer. In recent years, targeted inhibition of CDKs has shown considerable therapeutic benefit in a variety of tumor entities. Their success is reflected in clinical approvals of specific CDK4/6 inhibitors for breast cancer. This review provides a detailed insight into the molecular mechanisms of CDKs as well as a general overview of CDK inhibition. It also summarizes the latest research approaches and current advances in the treatment of head and neck cancer with CDK inhibitors. Instead of monotherapies, combination therapies with CDK inhibitors may especially provide promising results in tumor therapy. Indeed, recent studies have shown a synergistic effect of CDK inhibition together with chemo- and radio- and immunotherapy in cancer treatment to overcome tumor evasion, which may lead to a renaissance of CDK inhibitors.

The programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis blockade has been implemented in advanced-stage tumor therapy for various entities, including head and neck squamous cell carcinoma (HNSCC). Despite a promising tumor response in a subgroup of HNSCC patients, the majority suffer from disease progression. PD-L1 is known to influence several intrinsic mechanisms in cancer cells, such as proliferation, apoptosis, migration and invasion. Here, we modulated PD-L1 expression in three HNSCC cell lines with differential intrinsic PD-L1 expression. In addition to an alteration in the epithelial-to-mesenchymal transition (EMT) marker expression, we observed PD-L1-dependent cell spreading, migration and invasion in a spheroid spreading assay on four different coatings (poly-L-lysine, collagen type I, fibronectin and Matrigel^®) and a chemotactic transwell migration/invasion assay. Furthermore, the overexpression of PD-L1 led to increased gene expression and small interfering ribonucleic acid (siRNA) knockdown and decreased gene expression of Rho-GTPases and related proteins in a RT² Profiler™ PCR Array. Rac1 and Rho-GTPase pulldown assays revealed a change in the activation state concordantly with PD-L1 expression. In summary, our results suggest a major role for PD-L1 in favoring cell motility, including cell spreading, migration and invasion. This is presumably caused by altered N-cadherin expression and changes in the activation states of small Rho-GTPases Rho and Rac1.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytic, osteogenic, chondrogenic, and myogenic lineages. There is growing evidence that MSCs home into the tumor microenvironment attracted by a variety of signals such as chemokines, growth factors, and cytokines. Tumor‐homing stem cells may originate from bone marrow–derived MSCs (BMSCs) or adipose tissue‐derived MSCs. Recent scientific data suggest that MSCs in combination with tumor cells can either promote or inhibit tumorigenic behavior. In head and neck squamous cell carcinoma (HNSCC), BMSCs are reported to be enriched with a potential negative role. Here, we evaluated the effect of BMSCs from 4 different donors in combination with 4 HNSCC cell lines in a 3‐dimensional multicellular spheroid model. Heterogeneous combinations revealed an up‐regulation of gene and protein expression of osteogenic markers runt‐related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) together with a substantial secretion of matrix metalloproteinase 9. Moreover, heterogenous BMSC/tumor spheroids showed increased invasion compared with homogenous spheroids in a Boyden chamber invasion assay. Furthermore, inhibition of ALP resulted in a substantially decreased spreading of heterogeneous spheroids on laminin‐rich matrix. In summary, our data suggest a prometastatic effect of BMSCs combined with HNSCC.—Wessely, A., Waltera, A., Reichert, T. E., Stöckl, S., Grässel, S., Bauer, R. J. Induction of ALP and MMP9 activity facilitates invasive behavior in heterogeneous human BMSC and HNSCC 3D‐spheroids. FASEB J. 33, 11884–11893 (2019). www.fasebj.org

Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta

The aim of this study was to investigate the clinical, histopathologic, and immunologic differences of oral squamous cell carcinoma of never-smokers/never-drinkers and smokers/drinkers. Immunohistochemical staining for CD4, CD8, FoxP3, CD1a, and p16 was performed in 131 oral squamous cell carcinomas from smokers/drinkers and never-smokers/never-drinkers. Associations of smoking/drinking status with clinicopathologic data, immunohistochemical antibody expression, and survival were examined. Oral squamous cell carcinoma in never-smokers/never-drinkers was associated with the female gender (p < 0.001). Never-smokers/never-drinkers were older at diagnosis than smokers/drinkers (p < 0.001). Never-smokers/never-drinkers had more tumors in the maxilla, mandible, and tongue (p < 0.001). Pre-existing oral potentially malignant disorders appeared to be more common in never-smokers/never-drinkers (p < 0.001). Perineural invasion was more common in smokers/drinkers (p = 0.039). Never-smoking/never-drinking was associated with better overall survival (p = 0.004) and disease-specific survival (p = 0.029). High CD4+ T cell infiltration was associated with never-smoking/never-drinking (p = 0.008). Never-smokers/never-drinkers also showed increased CD8+ T cell infiltration (p = 0.001) and increased FoxP3+ Treg infiltration (p = 0.023). Furthermore, the total group of tumor-infiltrating lymphocytes was associated with never smoking/never drinking (p = 0.005). To conclude oral squamous cell carcinoma of the never-smokers/never-drinkers appears to be a distinct type of tumor, as it appears to have unique clinical and pathologic features and a more immunogenic microenvironment.

The programmed cell death 1 ligand 1 (PD‐L1)/programmed cell death protein 1 (PD‐1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD‐L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD‐L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD‐L1 in various cellular compartments of six well‐characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD‐L1 in its well‐known glycosylated/deglycosylated state at 40–55 kDa. In addition, we detected previously unknown PD‐L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD‐L1 variant expression is cell‐cycle‐dependent. Immunofluorescence staining of PD‐L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD‐L1 trafficking remain less understood; however, proximity ligation assays showed a cell‐cycle‐dependent interaction of the cytoskeletal protein vimentin with PD‐L1, whereas vimentin could serve as a potential shuttle for nuclear PD‐L1 transportation. Mass spectrometry after PD‐L1 co‐immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD‐L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD‐L1 and multiple tumor cell‐intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.