CTC together with Shh and Nrf2 are prospective diagnostic markers for HNSCC.

BACKGROUND: The lack of appropriate prognostic biomarkers remains a significant obstacle in the early detection of Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer type with a high mortality rate. Despite considerable advancements in treatment, the success in diagnosing HNSCC at an early stage still needs to be improved. Nuclear factor erythroid 2-related factor 2 (Nrf2) and Sonic Hedgehog (Shh) are overexpressed in various cancers, including HNSCC, and have recently been proposed as possible therapeutic targets for HNSCC. Circulating Tumor Cell (CTC) is a novel concept used for the early detection of cancers, and studies have suggested that a higher CTC count is associated with the aggressiveness of HNSCC and poor survival rates. Therefore, we aimed to establish molecular markers for the early diagnosis of HNSCC considering Shh/Nrf2 overexpression in the background. In addition, the relation between Shh/Nrf2 and CTCs is still unexplored in HNSCC patients. METHODS: In the present study, we selected a cohort of 151 HNSCC patients and categorized them as CTC positive or negative based on the presence or absence of CTCs in their peripheral blood. Data on demographic and clinicopathological features with the survival of the patients were analyzed to select the patient cohort to study Shh/Nrf2 expression. Shh and Nrf2 expression was measured by qRT-PCR. RESULTS: Considering significant demographic [smoking, betel leaf (p-value < 0.0001)] and clinicopathological risk factors [RBC count (p < 0.05), Platelet count (p < 0.05), Neutrophil count (p < 0.005), MCV (p < 0.0001), NLR (p < 0.05), MLR (p < 0.05)], patients who tested positive for CTC also exhibited significant overexpression of Shh/Nrf2 in both blood and tissue compared to CTC-negative patients. A strong association exists between CTCs and tumor grade. Following chemotherapy (a combination of Cisplatin, 5FU, and Paclitaxel), the frequency of CTCs was significantly decreased in patients with HNSCC who had tested positive for CTCs. The Kaplan-Meier plot illustrated that a higher number of CTCs is associated with poorer overall survival (OS) in patients with HNSCC. CONCLUSIONS: Detecting CTCs, and higher expression of Shh and Nrf2 in HNSCC patients' blood, can be a promising tool for diagnosing and prognosticating HNSCC.

Dual Effect of Organogermanium Compound THGP on RIG-I-Mediated Viral Sensing and Viral Replication during Influenza a Virus Infection.

The interaction of viral nucleic acid with protein factors is a crucial process for initiating viral polymerase-mediated viral genome replication while activating pattern recognition receptor (PRR)-mediated innate immune responses. It has previously been reported that a hydrolysate of Ge-132, 3-(trihydroxygermyl) propanoic acid (THGP), shows a modulatory effect on microbial infections, inflammation, and immune responses. However, the detailed mechanism by which THGP can modify these processes during viral infections remained unknown. Here, we show that THGP can specifically downregulate type I interferon (IFN) production in response to stimulation with a cytosolic RNA sensor RIG-I ligand 5'-triphosphate RNA (3pRNA) but not double-stranded RNA, DNA, or lipopolysaccharide. Consistently, treatment with THGP resulted in the dose-dependent suppression of type I IFN induction upon infections with influenza virus (IAV) and vesicular stomatitis virus, which are known to be mainly sensed by RIG-I. Mechanistically, THGP directly binds to the 5'-triphosphate moiety of viral RNA and competes with RIG-I-mediated recognition. Furthermore, we found that THGP can directly counteract the replication of IAV but not EMCV (encephalitismyocarditis virus), by inhibiting the interaction of viral polymerase with RNA genome. Finally, IAV RNA levels were significantly reduced in the lung tissues of THGP-treated mice when compared with untreated mice. These results suggest a possible therapeutic implication of THGP and show direct antiviral action, together with the suppressive activity of innate inflammation.

Chemotherapeutic resistance of head and neck squamous cell carcinoma is mediated by EpCAM induction driven by IL-6/p62 associated Nrf2-antioxidant pathway activation.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been associated with chemotherapeutic resistance, leads to aggressive tumor behavior, and results in an adverse clinical outcome. The molecular mechanism by which EpCAM enrichment is linked to therapeutic resistance via Nrf2, a key regulator of antioxidant genes is unknown. We have investigated the link between EpCAM and the Nrf2 pathway in light of therapeutic resistance using head and neck squamous cell carcinoma (HNSCC) patient tumor samples and cell lines. We report that EpCAM was highly expressed in Nrf2-positive and HPV-negative HNSCC cells. In addition, cisplatin-resistant tumor cells consisted of a higher proportion of EpCAMhigh cells compared to the cisplatin sensitive counterpart. EpCAMhigh populations exhibited resistance to cisplatin, a higher efficiency in colony formation, sphere growth and invasion capacity, and demonstrated reduced reactive oxygen species (ROS) activity. Furthermore, Nrf2 expression was significantly higher in EpCAMhigh populations. Mechanistically, expression of Nrf2 and its target genes were most prominently observed in EpCAMhigh populations. Silencing of EpCAM expression resulted in the attenuation of expressions of Nrf2 and SOD1 concomitant with a reduction of Sox2 expression. On the other hand, silencing of Nrf2 expression rendered EpCAMhigh populations sensitive to cisplatin treatment accompanied by the inhibition of colony formation, sphere formation, and invasion efficiency and increased ROS activity. The molecular mechanistic link between EpCAM expression and activation of Nrf2 was found to be a concerted interaction of interleukin-6 (IL-6) and p62. Silencing of p62 expression in EpCAMhigh populations resulted in the attenuation of Nrf2 pathway activation suggesting that Nrf2 pathway activation promoted resistance to cisplatin in EpCAMhigh populations. We propose that therapeutic targeting the Nrf2-EpCAM axis might be an excellent approach to modulate stress resistance and thereby survival of HNSCC patients enriched in EpCAMhigh populations.

Regulation of an adaptor protein STING by Hsp90ß to enhance innate immune responses against microbial infections.

Stimulator of interferon genes (STING) plays important roles in the DNA-mediated innate immune responses. However, the regulatory mechanism of STING in terms of stabilization is not fully understood. Here, we identified the chaperone protein Hsp90s as novel STING interacting proteins. Treatment with an Hsp90 inhibitor 17-AAG and knockdown of Hsp90ß but not Hsp90α reduced STING at protein level, resulted in the suppression of IFN induction in response to stimulation with cGAMP, and infections with HSV-1 and Listeria monocytogenes. Collectively, our results suggest that the control of STING protein by Hsp90ß is a critical biological process in the DNA sensing pathways.

Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18.

Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope design for the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out to predict the epitopes against HPV types 16 and 18 through "reverse vaccinology" approach. We attempted to identify the evolutionarily conserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within these regions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that the intertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. We had selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 of HPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA of L1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK, KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools for vaccine design against HPV.