Successful metastasis depends on cell invasion, migration, host immune escape, extravasation, and angiogenesis. The process of cell invasion and migration relies on the dynamic changes taking place in the cytoskeletal components; actin, tubulin and intermediate filaments. This is possible due to the plasticity of the cytoskeleton and coordinated action of all the three, is crucial for the process of metastasis from the primary site. Changes in cellular architecture by internal clues will affect the cell functions leading to the formation of different protrusions like lamellipodia, filopodia, and invadopodia that help in cell migration eventually leading to metastasis, which is life threatening than the formation of neoplasms. Understanding the signaling mechanisms involved, will give a better insight of the changes during metastasis, which will eventually help targeting proteins for treatment resulting in reduced mortality and longer survival.

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases encoded by 24 distinct genes. Their functions have been implicated in numerous normal and pathologic processes, including uterine involution and organogenesis, inflammation and wound healing, vascular and autoimmune disease progression. Pertinent to this review, the role of MMPs in cancer biology is fairly well researched and documented, and remains a subject of continuing intense investigation. Not only are several MMPs overexpressed in head and neck squamous cell carcinomas (HNSCCs), expression has been correlated with salient tumorigenic hallmarks, such as cell proliferation, angiogenesis, invasion, and metastasis. The utility of changes in the expression profile, as well as various MMP polymorphisms as potential prognostic markers in oral cancers and oral premalignant lesions, have been investigated. Furthermore, the potential therapeutic utility of targeting MMPs in cancer remains attractive, although outcomes in this respect appear so far to be less encouraging with respect to HNSCCs. Because of the disappointing results observed in clinical trials where MMP-targeting regimens for HNSCCs utilized broad-spectrum small MMP catalytic site inhibitors, investigators now envision new strategies for MMP-specific targeting based on the recognition of new noncatalytic MMP domains with distinct functions. This review provides an overview of MMP activities in general and in cancers, and an update of their activities in HNSCC. Specifically, their role in the development and progression of HNSCC and their function as signaling molecules is discussed. Finally, their role as potential prognostic biomarkers and therapeutic targets in HNSCC is revisited.

Dentin sialophosphoprotein (DSPP) is upregulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are commonly found under constant endoplasmic reticulum (ER) stress and exhibit increased levels of misfolded proteins, due to gene mutations and a stressful microenvironment. The present study examined the effects of DSPP silencing on the regulation of ER stress and the unfolded protein response (UPR) in oral cancer cells. A recently established stable DSPP short hairpin (sh)RNA-silenced OSC2 oral cancer cell line was used. The mRNA expression levels of ER stress-associated proteins, including 78 kDa glucose-regulated protein (GRP78), sarcoplasmic/endoplasmic reticulum calcium ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3r), protein kinase R-like endoplasmic reticulum kinase (PERK), serine/threonine-protein kinase/endoribonuclease IRE1 (IRE1), activating transcription factor 6 (ATF6) and matrix metalloproteinase 20 (MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B‑cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax) and cytochrome c] and cell proliferation-related [proliferating cell nuclear antigen (PCNA)] proteins were analyzed by western blotting. Cell viability, apoptosis and migration were monitored by MTT assay, Annexin V-fluorescein isothiocyanate flow cytometry and wound-healing assay, respectively. In transiently transfected puromycin‑free OSC2 cells, DSPP silencing markedly downregulated the mRNA expression levels of major ER stress regulators, including GRP78, SERCA2b, PERK, IRE1 and ATF6, as well as MMP20. DSPP silencing also resulted in decreased cell viability and migration, and enhanced apoptosis. Furthermore, PCNA and Bcl2 levels were decreased, whereas Bax and cytochrome c protein levels were increased in DSPP-silenced OSC2 cells. Sustained puromycin treatment partially counteracted the effects of DSPP silencing on the mRNA expression levels of ER stress-related proteins and MMP20, and on the migratory capacity of OSC2 cells. However, following puromycin treatment of DSPP-silenced cells, cell viability was further reduced and apoptosis was enhanced. In conclusion, these data provide evidence to suggest that DSPP may be involved in ER stress mechanisms in oral squamous cell carcinoma, since its downregulation in OSC2 cells led to significant alterations in the levels of major ER stress-associated proteins, and subsequent collapse of the UPR system.

BackgroundRecent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers.MethodsThe expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined.ResultsDSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 μM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%).ConclusionsWe conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.Electronic supplementary materialThe online version of this article (10.1186/s11658-018-0096-y) contains supplementary material, which is available to authorized users.

Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium.

Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP–MMP20 silencing on MMP2, MMP9, integrins αvβ3 and αvβ6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease (p < 0.05) in the expression of MMP2, MMP9, integrin αvβ3, αvβ6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased (p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).

Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.