Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer.

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for the development of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease9 (CRISPR/Cas9) gene editing technology that provided new tools for precise gene editing. It is possible to target any genomic locus virtually using only a complex nuclease protein with short RNA as a site-specific endonuclease. Since cancer is caused by genomic changes in tumor cells, CRISPR/Cas9 can be used in the field of cancer research to edit genomes for exploration of the mechanisms of tumorigenesis and development. In recent years, the CRISPR/Cas9 system has been increasingly used in cancer research and treatment and remarkable results have been achieved. In this review, we introduced the mechanism and development of the CRISPR/Cas9-based gene editing system. Furthermore, we summarized current applications of this technique for basic research, diagnosis and therapy of cancer. Moreover, the potential applications of CRISPR/Cas9 in new emerging hotspots of oncology research were discussed, and the challenges and future directions were highlighted.

The diagnostic value of GDF-15 for myocardial involvement in idiopathic inflammatory myopathy.

BACKGROUND: Cardiac involvement is a serious complication of idiopathic inflammatory myopathy (IIM). GDF-15 can predict the risk and the prognosis of cardiovascular disease, but its value is unclear in IIM. OBJECTIVE: To investigate the diagnostic value of GDF-15 for myocardial involvement in IIM. METHODS: A total of 77 IIM patients from May 2018 to August 2020 were included in this retrospective study. Of these, 43 patients underwent cardiac magnetic resonance (CMR) examination. There were 33 SLE patients and 16 healthy people were used as the control group. The concentration of GDF-15 of these groups was measured by ELISA. RESULTS: There were significant differences in GDF-15 levels in patients with IIM, SLE and healthy controls (H = 45.291, P<0.001). GDF-15 levels were statistically significant different between IIM patients with the myocardial injury [1484.88(809.07 2835.50) pg/ml] and without myocardial injury [593.26(418.61 784.59) pg/ml, P =0.001]. After adjusted for age, renal function, the risk of myocardial injury in IIM patients increased an average of 0.3% by per increased unit of GDF-15 (odds ratio=1.003, 95% CI: 1.000, 1.007). The level of GDF-15 was positively correlated with extra-cellular volume (ECV) (rs = 0.348, P =0.028). GDF-15 ≥ 929.505 pg/ml (area under the curve=0.856, 95% CI: 0.744, 0.968) predicted myocardial injury in IIM with a sensitivity of 0.75 and specificity of 0.90. CONCLUSION: GDF-15 could serve as a potential biomarker to predict myocardial injury in IIM patients.

PDK1 promotes metastasis by inducing epithelial-mesenchymal transition in hypopharyngeal carcinoma via the Notch1 signaling pathway.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare malignancy and has the worst prognosis among head and neck cancer. Metastasis is the major cause of poor prognosis in HSCC patients. In this study, we found that 3-phosphoinositide-dependent protein kinase 1 (PDK1 or PDPK1) was overexpressed in HSCC. The overexpression was positively correlated lymph node metastasis, clinical stage, and distant metastasis and indicated poor outcome. Loss and gain-of-function revealed that PDK1 increased cell proliferation, migration and invasion in vitro as well as tumor growth and metastasis in vivo. Mechanically, PDK1 induced epithelial-mesenchymal transition and promoted metastasis by activating the Notch1 signaling pathway. We further illustrated that PDK1 bound with the Notch1 intracellular domain, thereby inhibiting its ubiquitin-mediated degradation in a protein kinase B (Akt-) independent manner. In summary, PDK1/Notch1 axis played an important role in HSCC metastasis, and this investigation provided a new perspective on potential therapeutic targets for HSCC.

Epidemiology of pertussis in Alberta, Canada 2004-2015.

BACKGROUND: We describe the epidemiology of pertussis in Alberta, Canada by person, place, and time between 2004 and 2015, identify outbreak years, and examine vaccination coverage and vaccination timeliness. METHODS: We used health data from Alberta's Communicable Disease Registry System for the period of January 1, 2004 through August 31, 2015 to identify unique cases of pertussis. Unique cases were deterministically linked to data in Alberta's immunization repository and health care insurance plan registry. Population estimates and vaccination coverage were extracted from Alberta's online Interactive Health Data Application. We estimated pertussis incidence rates per 100,000 persons by year, age group, gender, and health zone. Outbreak years were identified using a one-sided cumulative sum (CUSUM) analysis by comparing annual incidence rates to baseline rates. RESULTS: Over the period, 3510 cases of pertussis were confirmed by laboratory testing or epidemiological linkage. Incidence rates per 100,000 persons were highest in 2004 (20.5), 2005 (13.6), and 2015 (10.4) for all age groups. Incidence rates were highest among the youngest age groups and decreased as age groups increased. Based on CUSUM analysis, 2008 and 2012 met the criteria for outbreak years. Vaccination coverage was over 90% among the general population, however only 61% of cases received at least one dose. About 60% of cases were diagnosed 5+ years after receiving the vaccine. Approximately 87-91% of vaccinated cases did not receive the first three vaccine doses in a timely manner. CONCLUSION: Pertussis incidence rates fluctuated over the period across all age groups. The majority of cases had no record of vaccination or were delayed in receiving vaccines. CUSUM analysis was an effective method for identifying outbreaks.

Triterpenoid Saponins with Potential Cytotoxic Activities from the Root Bark of Ilex rotunda Thunb.

A new 19-oxo-18,19-seco-ursane-type triterpeonoid saponin, laevigin E (8), together with 17 known compounds (1 - 7 and 9 - 18) were isolated from the root bark of Ilex rotunda Thunb. Their structures were determined by various spectroscopic analysis. Among them, compounds 6, 9, 11, and 18 were isolated from this species for the first time, while compounds 10 and 12 were firstly isolated from the family Aquifoliaceae. Biological activity assay showed that all triterpenoids exhibit moderate cytotoxic activities against MCF7, A549, HeLa and LN229 cell lines. The four triterpenoid saponins (3, 4, 6, and 8) exhibit slightly better activities compared to the four triterpenoid sapogenins (1, 2, 5, and 7). Compound 8 showed the best cytotoxicity against A549, HeLa and LN229 cell lines with IC50 of 17.83, 22.58 and 30.98 µm, respectively.

DDIT3 and KAT2A Proteins Regulate TNFRSF10A and TNFRSF10B Expression in Endoplasmic Reticulum Stress-mediated Apoptosis in Human Lung Cancer Cells.

TNFRSF10A and TNFRSF10B are cell surface receptors that bind to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediate the extrinsic pathway of apoptosis. However, the mechanisms of transcriptional regulation of TNFRSF10A and TNFRSF10B remain largely uncharacterized. In this study, two putative DDIT3 binding sites (-1636/-1625; -374/-364) and a putative AP-1 binding site (-304/-298) were identified in the TNFRSF10A promoter region. We found that DDIT3 interacts with phospho-JUN, and the DDIT3·phospho-JUN complex binds to the AP-1 binding site (-304/-298) within the TNFRSF10A promoter region. In addition, we confirmed that KAT2A physically interacts with the N-terminal region (amino acids 1-26) of DDIT3. Importantly, knockdown of KAT2A down-regulated TNFRSF10A and TNFRSF10B and dramatically decreased promoter activity of cells transfected with luciferase reporter plasmid containing the AP-1 binding site (-304/-298) of the TNFRSF10A promoter, as well as cells transfected with luciferase reporter plasmid containing DDIT3 binding site (-276/-264) of the TNFRSF10B promoter. ChIP results suggest that KAT2A may participate in a KAT2A·DDIT3·phospho-JUN complex, or may participate in a KAT2A·DDIT3 complex and acetylate H3K9/K14, respectively. Moreover, we verified that TNFRSF10A mediates apoptosis triggered by endoplasmic reticulum stress in human lung cancer cells. Collectively, we demonstrate that DDIT3 and KAT2A cooperatively up-regulate TNFRSF10A and TNFRSF10B. Our findings highlight novel mechanisms underlying endoplasmic reticulum stress-induced TNFRSF10A and TNFRSF10B expressions and apoptosis. These findings will be helpful for elucidating mechanisms related to anticancer drugs in mediating apoptosis.

A novel derivative of tetrandrine (H1) induces endoplasmic reticulum stress-mediated apoptosis and prosurvival autophagy in human non-small cell lung cancer cells.

H1, a bromized derivative of tetrandrine, has been reported to induce apoptosis in human cancer cells. But, the underlying mechanism of apoptosis triggered by H1 is unclear. In the present study, we found that H1 triggered death receptor 5 (DR5)-dependent apoptosis in non-small cell lung cancer (NSCLC) cells. Further study showed that H1 activated ER stress through enforcing the expression of Bip/GRP78, IRE1α, p-eIF2α, and CHOP. Moreover, abrogating CHOP expression blocked DR5 upregulation and subsequent apoptosis, indicating that CHOP was essential for DR5-dependent apoptosis induced by H1. In addition, H1 greatly downregulated cellular FLICE-inhibitory protein (c-FLIP), and enhanced expression of c-FLIP protected cancer cells from apoptosis in spite of H1 therapy. Furthermore, we discovered that H1 induced autophagy in human NSCLC cells. Interestingly, the autophagy induced by H1 played a protective function in NSCLC cells and effectively weakened caspase-mediated apoptosis. In summary, these findings suggest that H1 induces DR5-dependent apoptosis in human NSCLC cells via stimulating ER stress signaling pathway, and pharmacologically inhibiting autophagy will be an efficient approach to synergize H1-caused apoptosis in lung cancer cells.

MicroR-140-5p suppresses tumor cell migration and invasion by targeting ADAM10-mediated Notch1 signaling pathway in hypopharyngeal squamous cell carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that negatively regulate gene expression at the post-transcriptional level. Downexpression of miR-140-5p was reported in some human cancers, and combined with a reduction of cell migration and invasion, suggesting that miR-140-5p functions as a tumor suppressor. However, little is known about the expression and function of miR-140-5p in hypopharyngeal squamous cell carcinoma (HSCC). In this research, we found that miR-140-5p was significantly downregulated in HSCC tissues and correlated to tumor classification and lymph node metastasis. Restoration of miR-140-5p suppressed the migration and invasion of FaDu cells, and decreased the protein expression levels of ADAM10. Furthermore, the luciferase reporter assay revealed that miR-140-5p was directly bound to ADAM10 mRNA and knockdown of ADAM10 could inhibit FaDu cell migration and invasion and reduced the protein expression levels of and Notch1 intracellular domain (NICD1). Of note, knockdown of Notch1 could inhibit the migration and invasion of FaDu cells and rescued the effect of miR-140-5p inhibitor in FaDu cells. Taken together, our study demonstrates that miR-140-5p suppresses tumor migration and invasion by inhibiting ADAM10-mediated Notch1 signaling pathway and suggests that miR-140-5p could have potential therapeutic applications in HSCC.

HPV Vaccine utilization, Alberta 2008/09-2013/14 School year.

BACKGROUND: In Canada both bivalent (bHPV) vaccine and quadrivalent HPV vaccine (qHPV) are authorized for use. In Alberta, while both vaccines are available for private purchase, only qHPV is publicly funded for school girls in grades 5 and 9 as of 2013. We describe HPV vaccine uptake in Alberta, by school year, from the start of the publicly funded program in the Fall of 2008 through to August 31(st) 2014 and estimate the cumulative proportion of the female population who were vaccinated by the end of the 2013/14 school year. METHODS: We used data from the Alberta Ministry of Health Immunization and Adverse Reaction to Immunization repository (publicly funded vaccine), the population-based Pharmaceutical Information Network information systems (privately purchased vaccine) for the period September 1, 2008 to August 31, 2014 and demographic data from the Alberta Health Care Insurance Plan Registry. We estimate vaccine uptake rates and explore them by attributes of person, time, place, vaccine funding, and number of doses received. We estimated the cumulative proportions of the female population (by age group and number of doses received) who had received HPV vaccine by the end of the 2013/14 school year. RESULTS: Of the 169,259 unique individuals who received one or more doses of HPV vaccine over the period, 98.3% were females, and 83.8% received publicly funded vaccines. Vaccine uptake increased over the period. The cumulative proportion of females aged 9-26 years as of 2013/14 who had received two or more doses of vaccine was 34.3%; for those aged 10-11 years 59.6% and for those aged 14-15 years, 76.0%. For those aged 9-26 years, 31.3% had received three doses of vaccine. CONCLUSION: HPV vaccine uptake rates have increased in Alberta over the study period, most prominently among the age groups targeted by the publicly funded school-girl vaccine program.

A myrsinol diterpene isolated from Euphorbia prolifera reverses multidrug resistance in breast cancer cells.

P-glycoprotein (P-gp), a member of ATP-Binding Cassette transporter superfamily, can expel a variety of anti-cancer drugs so that it impairs the effect of cancer chemotherapy and results in multidrug resistance (MDR). The P-gp inhibitors are important to circumvent MDR and improve efficacy of cancer chemotherapy. The dried root of Euphorbia prolifera Buch.-Ham. has been used to treat cancer and inflammation in Chinese folk medicine for several hundred years. A myrsinol diterpene derived from Euphorbia prolifera Buch.-Ham. (J196-9-4) could modulate multidrug resistance. Cytotoxicity assays were performed to measure the reversal efficiency of J196-9-4. Efflux assay and ATPase assay were used to elucidate the mechanism of the chemical. J196-9-4 potentiated cytotoxicity of anti-cancer drugs in the P-gp over-expressing resistant breast cancer cell line MCF-7/Adr as compared to MCF-7 cells. Concentrations of 5 and 10 µM J196-9-4 could reverse the resistance to daunorubicin, vincristine, and topotecan significantly. Since J196-9-4 inhibited P-gp mediated efflux and stimulated ATP hydrolysis, J196-9-4 was a substrate of P-gp. Thus J196-9-4 is a competitive inhibitor of P-gp and reverses multidrug resistance induced by the transporter.

Chaetocin induces endoplasmic reticulum stress response and leads to death receptor 5-dependent apoptosis in human non-small cell lung cancer cells.

Epigenetic abnormalities are associated with non-small cell lung cancer (NSCLC) initiation and progression. Epigenetic drugs are being studied and in clinical trials. However, the molecular mechanism underlying the apoptosis by the epigenetic agents remains unclear. SUV39H1 is an important methyl-transferase for lysine 9 on histone H3 and usually related to gene transcriptional suppression, and chaetocin acts as the inhibitor of SUV39H1. We demonstrated here that chaetocin effectively suppressed the growth of multiple lung cancer cells through inducing apoptosis in a death receptor 5 (DR5)-dependent manner. Chaetocin treatment activated endoplasmic reticulum (ER) stress which gave rise to the up-regulation of ATF3 and CHOP. Furthermore, ATF3 and CHOP contributed to the induction of DR5 and subsequent apoptosis. When SUV39H1 was silenced with siRNA, the expression of ATF3, CHOP and DR5 was elevated. Thereafter, knockdown of SUV39H1 induced apoptosis in NSCLC cells. In summary, chaetocin pharmacologically inhibits the activity of SUV39H1 which provokes ER stress and results in up-regulation of ATF3 and CHOP, leading to DR5-dependent apoptosis eventually. These findings provide a novel interpretation on the anti-neoplastic activity of epigenetic drugs as a new therapeutic approach in NSCLC.

Work stress and alcohol consumption among adolescents: moderation by family and peer influences.

BACKGROUND: Excessive alcohol use in adolescence can be detrimental to health and academic performance. Few studies consider the moderating effects of parental and peer influence within the context of adolescent work outside of the school environment. This study aims to examine work stress among adolescents and the association with alcohol use and drunkenness, in the context of parental and peer influences. METHODS: Grade 12 students who participated in Monitoring the Future surveys between 2005 and 2009 (n = 12,341) were included in this study. Independent variables included work stress (job satisfaction, perceived safety, and perceived safety of possessions), self-reported perceptions towards academics and influence from parents and peers. Frequency of alcohol use and drunkenness were measured for lifetime, last 30 days and 12 months. The moderating effects of academic aspiration, parental, and peer influence were assessed on the relationship between work stress and alcohol use. RESULTS: Any work stress was positively associated with alcohol use over the past 12 months (odds ratio = 1.12, 95% confidence interval (CI) 1.02-1.23). Stratified analysis found that peer influence significantly moderated the relationship between work stress and alcohol use over the lifetime and past 12 months. Among adolescents with work stress, odds ratios of alcohol use over the lifetime was 0.83 (95% CI 0.71-0.97) for those with low negative peer influence and 1.09 (95% CI 0.97-1.22) for those with high negative peer influence. CONCLUSIONS: Problematic drinking patterns were more apparent among high school students who experienced stress at work. Positive peer influence, however, may buffer the adverse effect of work stress on alcohol use.

Herpes zoster vaccine (HZV): utilization and coverage 2009 - 2013, Alberta, Canada.

BACKGROUND: Herpes zoster vaccine (HZV) is not publicly funded in the province of Alberta, Canada. We estimated vaccine coverage among those aged 60 years or older for 2013, as well as vaccine utilization rates per hundred thousand population over the period 2009 - 2013. We explored for factors associated with HZV dispensing rates. METHODS: We used administrative data from the Alberta Pharmaceutical Information Network (PIN) database to identify unique persons for whom HZV had been dispensed from community pharmacies over 2009 - 2013. PIN data were also used to estimate the pharmacy/population ratios for rural and urban Alberta over the period. Denominators for rates were estimated using mid-year population estimates from the Alberta Health Care Insurance Plan Registry. Income quintile data were estimated from the 2006 Census of Canada. Crude, age, sex, geographic (rural vs. urban), income-quintile and year specific rates of HZV vaccine dispensing were estimated per 100,000 population. Rates were adjusted for pharmacy/population ratio. Vaccine coverage for persons aged 60 years or older was estimated using counts of all unique persons for whom the vaccine was dispensed over the period in the numerator and a 2013 mid- year population denominator. RESULTS: HZV dispensing rates rose annually from 2009 - 2013. Vaccine coverage was estimated to be 8.4% among persons aged 60 years or older. Rates of dispensing were highest for persons aged 60-69 years and were higher for females than males and for persons from higher compared to lower income quintiles. Dispensing rates were lower for rural than for urban residents. About 2% of vaccine was dispensed for persons aged less than 50 years. CONCLUSIONS: Rates of HZV dispensing are increasing rapidly in Alberta despite a lack of public funding. A small proportion of the vaccine may be dispensed off-label.

New proofs of some q-summation and q-transformation formulas.

We obtain an expectation formula and give the probabilistic proofs of some summation and transformation formulas of q-series based on our expectation formula. Although these formulas in themselves are not the probability results, the proofs given are based on probabilistic concepts.

[Molecular mapping of test mapping strain for 18th linkage group recessive genes elp, ch-2 and mln in silkworm (Bombyx mori)].

The ellipsoid egg, the second recessive gene of chocolate larvae, and melanism are controlled by three recessive genes, elp, ch-2, and mln in silkworm, respectively. Their order and genetic distance have been scheduled in established linkage group. Owing to lack of crossing over in females, the reciprocal backcrossed F1(BC1) progenies were bred for linkage analysis using the wild type silkworm strain P50(+elp+ch-2+mln /+elp+ch-2+mln) and W18 with ellipsoid egg, the second recessive gene of chocolate larvae, and melanism (elp ch-2 mln / elp ch-2 mln). In this research, we mapped three mutant genes, elp, ch-2, and mln on the chromosome 18 based on the SSR linkage map and STS markers designed based on silkworm genome sequence. The established linkage group, molecular linkage group, and the physic map of chromosome 18 had been corresponded. The genetic distance for this chromosome in this research was 94.2 cM, and the order of the mutants and molecular markers were consistent with the established silkworm linkage maps and the fine genome sequences. This research will lay important bases for map-based cloning for other mutants on chromosome 18.

The role of race/ethnicity in alcohol-attributable injury in the United States.

A substantial proportion of injuries worldwide are attributable to alcohol consumption, and US estimates indicate that the drinking patterns of racial/ethnic groups vary considerably. The authors reviewed evidence from 19 publications regarding racial/ethnic differences in overall alcohol-attributable injury as well as percent blood alcohol content positivity for injury deaths in the United States. They found that Native Americans evidence higher rates of alcohol-attributable motor vehicle crash fatality, suicide, and falls compared with other racial/ethnic groups; conversely, Asians evidence lower rates of alcohol-attributable injury than other racial/ethnic groups. The rate of alcohol positivity and intoxication among Hispanics is disproportionately high relative to estimates of alcohol use. Black subgroups also evidence higher rates of alcohol positivity than would be expected given estimates of alcohol use, including for alcohol positivity among drivers of fatally injured black children and homicide. These findings highlight the continued need for public health focus on Native American populations with respect to alcohol consumption and injury. Further, the disparity in alcohol-attributable injury mortality among black and Hispanic groups relative to their reported rates of alcohol consumption is an overlooked area of research. The authors review potential social determinants of racial/ethnic disparities in alcohol-attributable injuries and identify directions for further research on these patterns.

Claudin-1/4 as directly target gene of HIF-1α can feedback regulating HIF-1α by PI3K-AKT-mTOR and impact the proliferation of esophageal squamous cell though Rho GTPase and p-JNK pathway.

Immunohistochemical microarray comprising 80 patients with esophageal squamous cell carcinoma (ESCC) and discovered that the expression of CLDN1 and CLDN4 were significantly higher in cancer tissues compared to para-cancerous tissues. Furthermore, CLDN4 significantly affected the overall survival of cancer patients. When two ESCC cell lines (TE1, KYSE410) were exposed to hypoxia (0.1% O2), CLDN1/4 was shown to influence the occurrence and development of esophageal cancer. Compared with the control culture group, the cancer cells cultured under hypoxic conditions exhibited obvious changes in CLDN1 and CLDN4 expression at both the mRNA and protein levels. Through genetic intervention and Chip, we found that HIF-1α could directly regulate the expression of CLDN1 and CLDN4 in cancer cells. Hypoxia can affect the proliferation and apoptosis of cancer cells by regulating the PI3K-Akt-mTOR pathway. Molecular analysis further revealed that CLDN1 and CLDN4 can participate in the regulation process and had a feedback regulatory effect on HIF-1α expression in cancer cells. In vitro cellular experiments and vivo experiments in nude mice further revealed that changes in CLDN4 expression in cancer cells could affect the proliferation of cancer cells via regulation of Rho GTP and p-JNK pathway. Whether CLDN4 can be target for the treatment of ESCC needs further research.

Molecular Mechanisms of Cardiac Injury Associated With Myocardial SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread around the world. The development of cardiac injury is a common condition in patients with COVID-19, but the pathogenesis remains unclear. The RNA-Seq dataset (GSE150392) comparing expression profiling of mock human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and SARS-CoV-2-infected hiPSC-CMs was obtained from Gene Expression Omnibus (GEO). We identified 1,554 differentially expressed genes (DEGs) based on GSE150392. Gene set enrichment analysis (GSEA), Gene ontology (GO) analysis, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that immune-inflammatory responses were activated by SARS-CoV-2, while muscle contraction, cellular respiration, and cell cycle of hiPSC-CMs were inhibited. A total of 15 hub genes were identified according to protein-protein interaction (PPI), among which 11 upregulated genes were mainly involved in cytokine activation related to the excessive inflammatory response. Moreover, we identified potential drugs based on these hub genes. In conclusion, SARS-CoV-2 infection of cardiomyocytes caused a strong defensive response, leading to excessive immune inflammation, cell hypoxia, functional contractility reduction, and apoptosis, ultimately resulting in myocardial injury.

Afatinib induces pro-survival autophagy and increases sensitivity to apoptosis in stem-like HNSCC cells.

Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its antitumor effects in head and neck squamous cell carcinoma (HNSCC) by inducing intrinsic apoptosis through suppression of mTORC1. However, the detailed mechanism and biological significance of afatinib-induced autophagy in HNSCC remains unclear. In the present study, we demonstrated that afatinib induced mTORC1 suppression-mediated autophagy in HNSCC cells. Further mechanistic investigation revealed that afatinib stimulated REDD1-TSC1 signaling, giving rise to mTORC1 inactivation and subsequent autophagy. Moreover, ROS generation elicited by afatinib was responsible for the induction of the REDD1-TSC1-mTORC1 axis. In addition, pharmacological or genetic inhibition of autophagy sensitized HNSCC cells to afatinib-induced apoptosis, demonstrating that afatinib activated pro-survival autophagy in HNSCC cells. Importantly, in vitro and in vivo assays showed that afatinib caused enhanced apoptosis but weaker autophagy in stem-like HNSCC cells constructed by CDH1 knockdown. This suggested that blocking autophagy has the potential to serve as a promising strategy to target HNSCC stem cells. In conclusion, our findings suggested that the combination treatment with afatinib and autophagy inhibitors has the potential to eradicate HNSCC cells, especially cancer stem cells in clinical therapy.

MTDH in macrophages promotes the vasculogenic mimicry via VEGFA-165/Flt-1 signaling pathway in head and neck squamous cell carcinoma.

Vasculogenic mimicry (VM) refers to vessel-like structures formed by aggressive tumor cells and is closely associated with cancer invasion and metastasis. Here, we investigated the effect of macrophage-derived MTDH on VM formation in head and neck squamous cell carcinoma (HNSCC) and its underlying mechanism. Macrophages with MTDH overexpression (Mac-MTDH) promoted cancer cell VM formation, migration, and invasion in vitro. Moreover, MTDH overexpression triggered macrophage polarization into M2 type tumor-associated macrophages. Analysis of HNSCC clinical samples revealed that MTDH+ macrophages were predominantly located in the tumor-stromal region in proximity to VM and correlated with lymph node metastasis. Mechanistically, Mac-MTDH enhanced the expression and secretion of VEGFA-165 rather than other VEGFA isoforms via ß-catenin. The VEGFA-165/Flt-1 axis was responsible for Mac-MTDH's effects in cancer cells through p-STAT3/Twist1/VE-cadherin pathway. Using mouse model, we further confirmed that Mac-MTDH increased VM formation and cancer metastasis in vivo. Furthermore, in subcutaneous xenograft mouse model, HN6 + Mac-MTDH tumor exhibited elevated expression of p-STAT3 and Twist1 than HN6 + Mac-NC tumors. This study revealed that Mac-MTDH promoted VM formation, cancer cell migration and invasion, and cancer metastasis through VEGFA-165/Flt-1 axis, and that macrophage-derived MTDH could be a potential therapeutic target in HNSCC.