AB-free kava enhances resilience against the adverse health effects of tobacco smoke in mice.

Tobacco smoke remains a serious global issue, resulting in serious health complications, contributing to the onsets of numerous preventive diseases, and imposing significant financial burdens. Despite regulatory policies and cessation measures aimed at curbing its usage, novel interventions are urgently needed for effective damage reduction. Our preclinical and pilot clinical studies showed that AB-free kava has the potential to reduce tobacco smoke-induced lung cancer risk, mitigate tobacco dependence, and reduce tobacco use. To understand the scope of its benefits in damage reduction and potential limitations, this study evaluated the effects of AB-free kava on a panel of health indicators in mice exposed to 2 - 4 weeks of daily tobacco smoke exposure. Our comprehensive assessments included global transcriptional profiling of the lung and liver tissues, analysis of lung inflammation, evaluation of lung function, exploration of tobacco nicotine withdrawal, and characterization of the causal PKA signaling pathway. As expected, Tobacco smoke exposure perturbed a wide range of biological processes and compromised multiple functions in mice. Remarkably, AB-free kava demonstrated the ability to globally mitigate tobacco smoke-induced deficits at the molecular and functional levels with promising safety profiles, offering a unique promise to mitigate tobacco smoke-related health damages. Further pre-clinical evaluation and clinical translation are warranted to fully harness the potential of AB-free kava in combating tobacco smoke-related harms.

Florida-California Cancer Research, Education and Engagement (CaRE2) Health Equity Center: Structure, Innovations, and Initial Outcomes.

INTRODUCTION: The Florida-California Cancer Research, Education, and Engagement (CaRE2) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE2 triad partnership. METHODS: CaRE2 serves diverse populations in Florida and California using a "molecule to the community and back" model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. RESULTS: Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE2-related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. CONCLUSION: The CaRE2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Opportunities and Challenges of Kava in Lung Cancer Prevention.

Lung cancer is the leading cause of cancer-related deaths due to its high incidence, late diagnosis, and limited success in clinical treatment. Prevention therefore is critical to help improve lung cancer management. Although tobacco control and tobacco cessation are effective strategies for lung cancer prevention, the numbers of current and former smokers in the USA and globally are not expected to decrease significantly in the near future. Chemoprevention and interception are needed to help high-risk individuals reduce their lung cancer risk or delay lung cancer development. This article will review the epidemiological data, pre-clinical animal data, and limited clinical data that support the potential of kava in reducing human lung cancer risk via its holistic polypharmacological effects. To facilitate its future clinical translation, advanced knowledge is needed with respect to its mechanisms of action and the development of mechanism-based non-invasive biomarkers in addition to safety and efficacy in more clinically relevant animal models.

Lung microhaemorrhage drives oxidative/inflammatory damage in α1-antitrypsin deficiency.

Background: Animal models using intratracheal instillation show that elastase, unopposed by α1-antitrypsin (AAT), causes alveolar damage and haemorrhage associated with emphysematous changes. The aim of the present study was to characterise any relationship between alveolar haemorrhage and human AAT deficiency (AATD) using bronchoalveolar lavage (BAL) and lung explant samples from AATD subjects. Methods: BAL samples (17 patients, 15 controls) were evaluated for free haem (iron protoporphyrin IX) and total iron concentrations. Alveolar macrophage activation patterns were assessed using RNA sequencing and validated in vitro using haem-stimulated, monocyte-derived macrophages. Lung explants (seven patients, four controls) were assessed for iron sequestration protein expression patterns using Prussian blue stain and ferritin immunohistochemistry, as well as ferritin iron imaging and elemental analysis by transmission electron microscopy. Tissue oxidative damage was assessed using 8-hydroxy-2'-deoxyguanosine immunohistochemistry. Results: BAL collected from AATD patients showed significantly elevated free haem and total iron concentrations. Alveolar and interstitial macrophages in AATD explants showed elevated iron and ferritin accumulation in large lysosomes packed by iron oxide cores with degraded ferritin protein cages. BAL macrophage RNA sequencing showed innate pro-inflammatory activation, replicated in vitro by haemin exposure, which also triggered reactive oxygen species generation. AATD explants showed massive oxidative DNA damage in both lung epithelial cells and macrophages. Conclusions: BAL and tissue markers of alveolar haemorrhage, together with molecular and cellular evidence of macrophage innate pro-inflammatory activation and oxidative damage, are consistent with free haem stimulation. Overall, this initial study provides evidence for a pathogenetic role of elastase-induced alveolar haemorrhage in AATD emphysema.

Investigating miR-9 as a mediator in laryngeal cancer health disparities.

Background: For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Methods: Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Results: Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Conclusion: Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

The Intersectionality between Race, Ethnicity, and Residential-Level Socioeconomic Status in Disparities of Head and Neck Cancer Outcomes: A SEER Study.

BACKGROUND: Head and neck cancer (HNC) mortality differs by race, ethnicity, and socioeconomic status (SES). However, it is unclear whether the relationship between race/ethnicity and HNC-specific mortality varies according to the residence-level SES. METHODS: Data from the Surveillance Epidemiology and End Results database included participants with primary HNC between 2006 and 2017 (followed through 2018) to assess the joint association of race/ethnicity and census-tract level SES Yost-index groups (quintiles) with all-cause and HNC-specific mortalities. Relative survival rates at 1, 5, and 10 years were calculated. Multivariable Cox proportional hazard regression models estimated hazard-ratios and 95% confidence intervals for all-cause mortality, and Fine-Gray subdistribution hazard models for HNC-specific mortality. Cumulative incidence curves for HNC-specific deaths were estimated. RESULTS: 76,095 patients were included in the analysis: 63.2% were <65 years, 73.4% male, and 11.3% non-Hispanic (NH) Black. Most patients (58.3%) were diagnosed at regional or distant stages and 20.6% died of HNC. The five-year relative survival rate increased with SES group, with 51.6% in the lowest SES group, and 74.1% in the highest SES group. NH-Black patients had higher risk of all-cause and HNC-specific mortality than NH-White patients, regardless of the SES group. NH-Asian/Pacific Islander and Hispanic patients had higher risk of HNC-specific mortality in some SES groups. CONCLUSIONS: NH-Black patients of all SES strata had significantly worse outcomes. Other factors, such as healthcare quality, may be associated with persistent disparities. IMPACT: The study highlights the persistence of significant racial disparities in HNC survival across socioeconomic categories. There is need to consider additional factors underlying these disparities.

A 40-Year-Old Woman With COVID-19 and Bilateral Vision Loss.

Mucormycosis is a fast-spreading angioinvasive fungal infection with a very high mortality rate. It is associated with immunodeficiency, diabetes mellitus, iron overload, stem cell transplantation and the use of steroids. As cultures and histopathological biopsy may have low yield in invasive fungal infections, new generation sequencing of cfDNA (cell free deoxyribonucleic acid) has become a cornerstone for diagnosis. Over the past 18 months, increasing reports of COVID-19 associated Mucormycosis have emerged, most specifically in India and other nearby developing countries. Awareness and knowledge of this newly discovered association is of high importance and clinical relevance as the global COVID-19 pandemic continues. Herein, we present a case of a patient who was treated with steroids for COVID-19 in the outpatient setting and presented with unilateral periorbital pain and blurry vision. She progressively developed bilateral vision loss, fixed bilateral mydriasis, ophthalmoplegia and coma. Imaging findings included leptomeningeal, vascular, and subcortical enhancement accompanied with multifocal infarction. Subsequent biopsy of the paranasal sinuses revealed broad type fungal elements and cfDNA sequencing identified the pathogen as Rhizopus species. She was treated with intravenous amphotericin B, but succumbed to the infection.

Cell Dissociation Enzymes Affect Annexin V/Flow-Cytometric Apoptotic Assay Outcomes After miRNA-based Transient Transfection.

BACKGROUND/AIM: miRNA functional analysis involves transfection with miRNA-based oligos to identify gain-of or loss-of function cellular phenotypes. Apoptosis is a common phenotypic endpoint for miRNA functional analysis. We report that four common cell dissociation enzymes, TrypLE, Accutase, Trypsin, and Accumax, can differentially impact cell viability and apoptosis in Annexin V flow cytometric analysis after miRNA-based transient transfection. MATERIALS AND METHODS: We transiently transfected a nonsense oligo into an epithelial cancer cell line (UM-SCC-12) for 24 h. Cells were harvested with either TrypLE, Accutase, Accumax, or Trypsin after 5 min. The Annexin V/7-AAD assay via flow cytometry was employed. Studies were performed in triplicate. Significant effects were detected by ANOVA, followed by Tukey's Multiple Comparison tests. RESULTS: Trypsin produced the lowest cell viability and lowest percentage of apoptotic cells, specifically when compared to TrypLE and Accutase, respectively (p<0.01). Importantly, transfected trypsinized cells had a significant difference in cell viability and necrosis (p<0.05) when compared with non-transfected trypsinized cells, highlighting the influence of miRNA-based transfection on Annexin V flow cytometric outcomes. Interassay variability was lowest with TrypLE (1.13 %). As such, TrypLE provided the greatest reproducibility and reliability in our cell line. CONCLUSION: Our study highlights the variable effects of cell dissociation enzymes on transfected cells. Overall, the variability may lead to errors in detection of apoptotic cells using the Annexin V assay after miRNA-based transfection. Before assay use, we recommend pretesting cell dissociation enzymes on transfected cells to ensure reliable and reproducible results.

Lower disease control rates and survival outcomes among Blacks with pharyngeal squamous cell carcinomas compared with Whites: a retrospective analysis at the University of Florida.

Disparate clinical outcomes for pharyngeal squamous cell carcinoma (PSCC) of the oropharynx (OPSCC) and hypopharynx (HPSCC) have been observed in Black compared with White patients. Higher tobacco and alcohol use has been associated with decreased survival in Black patients with PSCC. Higher human papilloma virus (HPV) infection rates, associated with specific subsites of the oropharynx, are linked to improved overall survival (OS). Using an institutional cohort of Black and White patients with PSCC, we performed a retrospective analysis using multiple disease endpoints including local control (LC), local-regional control (LRC), freedom from distant metastases (DMFS), OS, cause-specific survival (CSS), and recorded tobacco and alcohol use. 1419 patients [Black (n = 111) and White (n = 1,308)] treated for PSCC from 1973 to 2013 were evaluated. PSCC 5- and 10-year LC, LRC, and DMFS and CSS rates were lower for Blacks. Notably, Black patients with OPSCC had higher stage cancers, higher percentage of soft palate tumors, and lower percentage of base of tongue cancers, were more likely to receive radiotherapy, and had higher tobacco and alcohol use. OS was significantly lower in Black patients at both anatomic sites, with the greatest difference observed for OPSCC. Multivariate analysis showed race and tobacco independently predicted DMFS, OS, and CSS; however, tobacco use had a greater impact on DMFS (HR 2.5, p = 0.021) than race (HR 1.9, p = 0.027). Overall, we propose that the higher burden of tobacco use along with a lower rate of tumors arising from traditional HPV-related subsites were important contributors to disparate disease outcomes seen in our Black patients.

Loss of RE-1 silencing transcription factor accelerates exocrine damage from pancreatic injury.

Regulation of pancreas plasticity is critical for preventing injury and promoting regeneration upon tissue damage. The intricate process of pancreatic differentiation is governed by an orchestrated network of positive and negative transcription factors for appropriate gene expression. While the transcriptional repressor REST is well characterized as a silencer of neuronal genes in non-neuronal cells, the role of REST in regulating exocrine pancreas cell identity remains largely unexplored. Rest expression is increased upon injury in the mouse pancreas, such as induced acute and chronic pancreatitis and ductal adenocarcinoma. At the cellular level, Rest expression is lower in mature acinar cells compared with pancreas progenitor and ductal cells. To investigate the role of REST activity in pancreatic transdifferentiation and homeostasis, we developed a novel mouse model (Cre/RESTfl/fl) with conditional knockout (KO) of Rest expression within pancreas cells. The high Cre-mediated excision efficiency of Rest exon two KO caused decreased Rest expression and activity within the pancreas. Short-term organoid cultures of pancreatic acini to undergo acinar-to-ductal metaplasia (ADM) showed that loss of REST impedes induced ADM, while overexpression of REST increases ADM. Interestingly, REST ablation accelerated acute pancreatitis in mice treated with the cholecystokinin analog caerulein, as indicated by cellular morphology, elevated serum amylase levels and pancreatic edema. Furthermore, Cre/RESTfl/fl mice were more sensitive to acute pancreatitis injury and displayed augmented tissue damage and cellular lesions. These results suggest REST has a novel protective role against pancreatic tissue damage by acting as a regulator of exocrine cell identity.

MYB-activated models for testing therapeutic agents in adenoid cystic carcinoma.

OBJECTIVE: There are no effective systemic therapies for adenoid cystic cancer (ACC) and lack of tumor lines and mouse models have hindered drug development.We aim to develop MYB-activated models for testing new therapeutic agents. MATERIALS AND METHODS: We studied new ACC patient-derived xenograft (PDX) models and generated a matched cell line from one patient. In addition, we generated a genetically-engineered MYB-NFIB mouse model (GEMM) that was crossed with Ink4a+/-/Arf+/- mice to study tumor spectrum and obtain tumor lines. Using human and murine ACC-like tumor lines, we analyzed MYB expression by RNA-Seq and immunoblot and tested efficacy of new MYB inhibitors. RESULTS: We detected MYB-NFIB transcripts in both UFH1 and UFH2 PDX and observed tumor inhibition by MYB depletion using shRNA in vivo. We observed rapid loss of MYB expression when we cultured UFH1 in vitro, but were able to generate a UFH2 tumor cell line that retained MYB expression for 6 months. RNA-Seq expression detected an ACC-like mRNA signature in PDX samples and we confirmed an identical KMT2A/MLL variant in UFH2 PDX, matched cell line, and primary biopsy. Although the predominant phenotype of the MYB-NFIB GEMM was B-cell leukemia, we also generated a MYB-activated ACC-like mammary tumor cell line. We observed tumor inhibition using a novel MYB peptidomimetic in both human and murine tumor models. CONCLUSIONS: We generated and studied new murine and human MYB-activated tumor samples and detected growth inhibition with MYB peptidomimetics. These data provide tools to define treatment strategies for patients with advanced MYB-activated ACC.

CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma.

OBJECTIVES: In accordance with the Precision Medicine Initiative, new treatment strategies for head and neck squamous cell carcinoma (HNSCC) are needed to yield better therapeutic outcomes. The purpose of this study was to establish and validate chimeric antigen receptor (CAR)-T cells targets in HNSCC. METHODS: Putative CAR-T antigens were identified in The Cancer Genome Atlas database. To validate antigen suitability, quantitative RT-PCR, flow cytometry, and immunofluorescent staining were performed. A retroviral human CD70 CAR construct, using truncated CD27 conjugated with 4-1BB and CD3-zeta costimulatory molecules, was used to transduce activated human T cells to generate CD70 CAR-T cells. Cell-based cytotoxicity and cytokine ELISAs were used to measure efficacy of killing. RESULTS: Nine potential CAR-T targets (CD276, EGFR, MICA, MICB, MAGE-A4, FAP, EPCAM, CD70, B4GALNT1) were identified based on their high expression in tumors compared to flanking control tissues. CD70 was selected for further proof-of-principle analysis based on its differential expression in several tumor subtypes, and showed substantial heterogeneity in individual tumors analyzed. Cell surface CD70 protein and CD70 mRNA were detected from low to high levels in established HNSCC cancer cell lines. CD70 was highly expressed in 4 of 21 tumor biopsies (19%), and 3 of 4 specimens showed strong CD70 expression on the tumor cell surface. CD70-specific CAR-T cells were generated and further demonstrated to recognize and kill CD70-positive HNSCC cells efficiently, but not CD70-negative cancer cells. CONCLUSION: CD70-specific CAR-T cells specifically recognized and efficiently eliminated CD70-positive HNSCC cells. This study provides the basis for further investigation into CD70 and other CAR-T targets.

Primary orbital melanoma treated with orbital exenteration and postoperative radiotherapy: A case report and review of the literature.

BACKGROUND: We report on a patient with human immunodeficiency virus (HIV)-positive disease with a primary orbital melanoma treated with surgery and adjuvant radiation. METHODS: A 53-year-old woman with HIV-positive disease presented with left-sided progressive ipsilateral vision loss and proptosis. An MRI scan revealed a mass-enhancing lesion measuring 2.1 × 2.6 × 2.5 cm abutting the optic nerve. The patient underwent left orbital exenteration with temporalis flap reconstruction, pathology revealing malignant melanoma, stage T1N0M0. Posterior margins were positive and lymphovascular invasion was present; therefore, the patient received adjuvant radiation to a total dose of 70 Gy in 35 fractions. RESULTS: The patient remains with no evidence of disease (NED) at a follow-up time of 3.5 years. CONCLUSION: Surgery remains the mainstay of treatment in patients with primary orbital melanomas, and adjuvant radiotherapy should be considered for those with positive margins or other risk factors for recurrence. We present a patient with significant risk factors with NED at 3.5-year follow-up.

Eps8 expression is significantly lower in p16+ head and neck squamous cell carcinomas (HNSCCs) compared with p16- HNSCCs.

In vitro head and neck cancer studies have demonstrated that epidermal growth factor receptor kinase substrate 8 (Eps8) overexpression contributes to squamous carcinogenesis. Oral squamous cell carcinoma studies have correlated Eps8 expression with metastatic disease and poor prognosis. Head and neck squamous cell carcinoma (HNSCC) studies comparing its expression by anatomic site or in in vivo regional metastases have not been performed. In this study, we compared Eps8 expression in HNSCCs arising in the oral cavity (OCSCC) and oropharynx (OPSCC) along with their corresponding regional lymph node (LN) metastases. We then correlated our findings with clinicopathologic data including tumor-node-metastasis stage, p16 status, age, sex, and smoking and alcohol history. Eps8 immunohistochemistry was performed on 69 archived OCSCCs and OPSCCs, and 24 paired and 4 unpaired LNs. Expression was scored from 0 to 3. Eps8 expression was detected in 49% of combined OCSCC and OPSCC cases. We found that expression correlated with advanced tumor stage (P = .022) and p16 status (P = .032) but not with anatomic site. Notably, p16+ HNSCCs had significantly lower Eps8 expression than p16- HNSCCs. No significant difference was observed between primary HNSCCs and their corresponding metastatic LNs. Neither p16 status nor anatomic site influenced Eps8 expression in regional LN metastases. In conclusion, our data offer in vivo support that, in HNSCCs, Eps8 is involved in tumor invasion but not necessarily the development of regional LN metastasis. The association between low Eps8 expression and p16+ HNSCCs suggests that alternative signaling pathways may be used for their tumorigenesis.