KEAP1/NRF2 Mutations in Stem Cells Define an Aggressive Subset of Head and Neck Cancer Patients Who Have a Poor Prognosis, Lung Metastasis, and Therapeutic Failure.

Mutations in Keap1/Nrf2 in head and neck cancer result in abnormal cell growth. Progenitor cells, bulk tumor cells, and head and neck cancer stem cells (HN-CSCs) may all harbor these mutations. Nevertheless, whether Keap1/Nrf2 mutations in HN-CSCs have an impact on clinical outcomes is unknown. Cancerous HN-CSCs and benign stem cells were obtained from freshly resected head and neck cancer patients (n = 50) via flow cytometry cell sorting and tested for Keap1/Nrf2 mutations. The existence of Keap1/Nrf2 mutations in HN-CSCs, as well as their correlations with tumor mutations, pathologic tumor stage, tumor histologic grades, lung metastasis, treatment outcomes, and the patient's age and conditions, are assessed at the last follow-up visit. Thirteen tumors were found to have Keap1/Nrf2 mutations in their HN-CSCs. More than half of the lung metastases and disease progression occurred in HN-CSCs with mutations. Patients whose tumors carried Keap1/Nrf2 mutations in their HN-CSCs had significantly shorter progression-free survival, overall survival, and time of treatment failure than their non-HN-CSC counterparts. These associations were partly driven by HN-CSCs, in which Keap1/Nrf2 mutations were overrepresented in fast progressors and associated with an increased risk of disease progression. Our findings suggest that molecular genotyping of HN-CSCs may facilitate personalized treatment strategies and assist in identifying patients who are likely to benefit from chemotherapy.

The metastatic capacity of high-grade serous ovarian cancer cells changes along disease progression: inhibition by mifepristone.

BACKGROUND: Simplistic two-dimensional (2D) in vitro assays have long been the standard for studying the metastatic abilities of cancer cells. However, tri-dimensional (3D) organotypic models provide a more complex environment, closer to that seen in patients, and thereby provide a more accurate representation of their true capabilities. Our laboratory has previously shown that the antiprogestin and antiglucocorticoid mifepristone can reduce the growth, adhesion, migration, and invasion of various aggressive cancer cells assessed using 2D assays. In this study, we characterize the metastatic capabilities of high-grade serous ovarian cancer cells generated along disease progression, in both 2D and 3D assays, and the ability of cytostatic doses of mifepristone to inhibit them. METHODS: High-grade serous ovarian cancer cells collected from two separate patients at different stages of their disease were used throughout the study. The 2D wound healing and Boyden chamber assays were used to study migration, while a layer of extracellular matrix was added to the Boyden chamber to study invasion. A 3D organotypic model, composed of fibroblasts embedded in collagen I and topped with a monolayer of mesothelial cells was used to further study cancer cell adhesion and mesothelial displacement. All assays were studied in cells, which were originally harvested from two patients at different stages of disease progression, in the absence or presence of cytostatic doses of mifepristone. RESULTS: 2D in vitro assays demonstrated that the migration and invasive rates of the cells isolated from both patients decreased along disease progression. Conversely, in both patients, cells representing late-stage disease demonstrated a higher adhesion capacity to the 3D organotypic model than those representing an early-stage disease. This adhesive behavior is associated with the in vivo tumor capacity of the cells. Regardless of these differences in adhesive, migratory, and invasive behavior among the experimental protocols used, cytostatic doses of mifepristone were able to inhibit the adhesion, migration, and invasion rates of all cells studied, regardless of their basal capabilities over simplistic or organotypic metastatic in vitro model systems. Finally, we demonstrate that when cells acquire the capacity to grow spontaneously as spheroids, they do attach to a 3D organotypic model system when pre-incubated with conditioned media. Of relevance, mifepristone was able to cause dissociation of these multicellular structures. CONCLUSION: Differences in cellular behaviours were observed between 2 and 3D assays when studying the metastatic capabilities of high-grade serous ovarian cancer cells representing disease progression. Mifepristone inhibited these metastatic capabilities in all assays studied.

Genetic alterations of Keap1 confers chemotherapeutic resistance through functional activation of Nrf2 and Notch pathway in head and neck squamous cell carcinoma.

Keap1 mutations regulate Nrf2 activity and lead to chemoresistance in cancers. Yet the underlying molecular mechanisms of chemoresistance are poorly explored. By focusing and genotyping head and neck squamous cell carcinoma (HNSCC) that had available pathologic and clinical data, we provide evidence that Keap1 displays frequent alterations (17%) in HNSCC. Functional loss of Keap1 results in significant activation of Nrf2 and promotes cancer cell growth, proliferation, and elevated cancer stem cell (CSCs) self-renewal efficiency and resistance to oxidative stress. Furthermore, decreased Keap1 activity in these cells increased nuclear accumulation of Nrf2 and activation of the Notch pathway, causing enhanced transcriptional alterations of antioxidants, xenobiotic metabolism enzymes, and resistance to chemotherapeutic treatment. Limiting the Nrf2 activity by either Keap1 complementation or by Nrf2 silencing increased the sensitivity to chemotherapy in Keap1-mutated cells and repressed the CSC self-renewal activity. Our findings suggest that Keap1 mutations define a distinct disease phenotype and the Keap1-Nrf2 pathway is one of the leading molecular mechanisms for clinical chemotherapeutic resistance. Targeting this pathway may provide a potential and attractive personalized treatment strategy for overcoming chemotherapeutic resistance conferred by Keap1 mutations.

Decline in Respiratory Functions in Hospitalized SARS-CoV-2 Infected Cancer Patients Following Cytotoxic Chemotherapy-An Additional Risk for Post-chemotherapy Complications.

Background: Patients recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection demonstrate impaired lung function and those requiring chemotherapy after recovering from SARS-CoV-2 infection have yet to be explored. In this study, we sought to investigate the possible pulmonary functional changes during and after administering chemotherapy in patients with prior SARS-CoV-2 infection. Methods: In this study, a total of 37 SARS-CoV-2 infected patients with cancer who were discharged from hospital and received subsequent cytotoxic chemotherapy were enrolled and prospectively followed-up. The following parameters were prospectively measured before (P1), after first chemotherapy cycle (P2), and 10 weeks after the end of chemotherapy (P3), to assess their impact on respiratory complications in terms of diffusion capacity of the lungs for carbon monoxide (DLCO), forced expiratory volume in 1-s (FEV1), forced vital capacity (FVC), 6-min walking distance (6MWD) test and levels of key inflammatory markers. Results: All patients completed at least 2 cycles of chemotherapy without showing overt respiratory complications. Six patients (16%) complained about dyspnea during chemotherapy or at follow-up period. DLCO was significantly impaired during follow-up period [from P1 78 to P3 60% of predicted values; interquartile range (IQR) 55-89] and in 32 of 37 (86% of patients) from P1 to P2 (65% of predictive value; IQR 58-70; p < 0.001). Several patients experienced post-chemotherapy respiratory complications. As expected, all patients from control groups showed persistent improved pulmonary functions. Conclusion: The risk of pulmonary impairments due to cytotoxic chemotherapy in prior SARS-CoV-2 infected patients is linked to the loss of DLCO. Accordingly, we recommend that for patients with cancer requiring chemotherapy after recovering from prior SARS-CoV-2 infection, pulmonary tests to be performed routinely before and during chemotherapy treatment to monitor the pulmonary performance.

SARS-CoV-2 Viral Load Is Correlated With the Disease Severity and Mortality in Patients With Cancer.

The correlation between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load and risk of disease severity in cancer patients is poorly understood. Given the fact that cancer patients are at increased risk of severe coronavirus disease 2019 (COVID-19), analysis of viral load and disease outcome in COVID-19-infected cancer patients is needed. Here, we measured the SARS-CoV-2 viral load using qPCR cycle threshold (Ct) values collected from 120 noncancer and 64 cancer patients' nasopharyngeal swab samples who are admitted to hospitals. Our results showed that the in-hospital mortality for high viral load cancer patients was 41.38%, 23.81% for medium viral load and 14.29% for low viral load patients (p < -0.01). On the other hand, the mortality rate for noncancer patients was lower: 22.22% among patients with high viral load, 5.13% among patients with medium viral load, and 1.85% among patients with low viral load (p < 0.05). In addition, patients with lung and hematologic cancer showed higher possibilities of severe events in proportion to high viral load. Higher attributable mortality and severity were directly proportional to high viral load particularly in patients who are receiving anticancer treatment. Importantly, we found that the incubation period and serial interval time is shorter in cancer patients compared with noncancer cases. Our report suggests that high SARS-CoV-2 viral loads may play a significant role in the overall mortality and severity of COVID-19-positive cancer patients, and this warrants further study to explore the disease pathogenesis and their use as prognostic tools.

Radioresistance in Glioblastoma and the Development of Radiosensitizers.

Ionizing radiation is a common and effective therapeutic option for the treatment of glioblastoma (GBM). Unfortunately, some GBMs are relatively radioresistant and patients have worse outcomes after radiation treatment. The mechanisms underlying intrinsic radioresistance in GBM has been rigorously investigated over the past several years, but the complex interaction of the cellular molecules and signaling pathways involved in radioresistance remains incompletely defined. A clinically effective radiosensitizer that overcomes radioresistance has yet to be identified. In this review, we discuss the current status of radiation treatment in GBM, including advances in imaging techniques that have facilitated more accurate diagnosis, and the identified mechanisms of GBM radioresistance. In addition, we provide a summary of the candidate GBM radiosensitizers being investigated, including an update of subjects enrolled in clinical trials. Overall, this review highlights the importance of understanding the mechanisms of GBM radioresistance to facilitate the development of effective radiosensitizers.

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.

Widespread expression of Sonic hedgehog (Shh) and Nrf2 in patients treated with cisplatin predicts outcome in resected tumors and are potential therapeutic targets for HPV-negative head and neck cancer.

BACKGROUND: Sonic hedgehog (Shh) and Nrf2 play a critical role in chemotherapeutic resistance. These two genes have been found to be dysregulated in head and neck squamous cell carcinomas (HNSCC). The purpose of this study was to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs). METHODS: We analyzed a cohort of patients with HNSCC to identify potential therapeutic biomarkers correlating with overall survival (OS) as well as disease-free survival (DFS) from our own data and validated these results using The Cancer Genome Atlas dataset. Expression of Shh and Nrf2 was knocked down by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated. RESULTS: Widespread abundant expression of Shh and Nrf2 proteins were associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a significant predictor of patient DFS. The tumor stromal index was correlated with Shh expression and inversely associated with shorter OS and DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to the chemotherapeutic agents. Concomitant upregulation of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in patients with HNSCC. CONCLUSIONS: These findings suggest that Shh and Nrf2 could serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC.

Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1.

BACKGROUND: A newly developed drug trastuzumab emtansine (T-DM1) has improved the survival of breast cancer (BC) patients. Despite an impressive initial clinical response, a subgroup of patient develop resistance and present therapeutic challenges. The underlying resistance mechanisms are not fully investigated. We report that T-DM1 treatment modulates the expression of ROR1 (type 1 receptor tyrosine kinase-like orphan receptor) and induces self-renewal of cancer stem cells (CSCs) leading to therapeutic resistance. METHODS: Using BC patient tumor samples, and BC cell lines we gained insight into the T-DM1 treatment induced ROR1 overexpression and resistance. In vitro sphere forming assays and in vivo extreme dilution assays were employed to analyze the stemness and self-renewal capacity of the cells. A series of molecular expression and protein assays including qRT-PCR, FACS-sorting, ELISA, immunostaining, Western blotting were used to provide evidence. FINDINGS: Exposure of cells to T-DM1 shifted ROR1 expression from low to high, enriched within the CSC subpopulation, coincident with increased Bmi1 and stemness factors. T-DM1 induced ROR1 cells showed high spheroid and tumor forming efficiency in vitro and in an animal model exhibiting shorter tumor-free time. Mechanistically, the overexpression of ROR1 is partly induced by the activation of YAP1 and its target genes. Silencing of ROR1 and YAP1 by pharmacologic inhibitors and/or sh/siRNA inhibited spheroid formation, the initiation of tumors and the capacity for self-renewal and ROR1 overexpression. INTERPRETATIONS: The results presented here indicate that simultaneous targeting of ROR1 and YAP1 may suppress CSC self-renewal efficacy and inhibit tumor progression in BC. In this manner such treatments may overcome the T-DM1 mediated therapeutic resistance and improve clinical outcome. FUND: This study was supported by Neurogen Technologies for interdisciplinary research.

Chaperone gp96-independent inhibition of endotoxin response by chaperone-based peptide inhibitors.

HSP90 chaperones a large number of proteins, and it plays essential roles in multiple signaling pathways to maintain protein homeostasis in the cytosol. In addition, HSP90 has been implicated in mediating recognition of lipopolysaccharide (LPS). However, no pharmacologic agents have been developed to interrogate this pathway. Herein we demonstrate that a peptide-based inhibitor that was previously reported to inhibit the master Toll-like receptor-chaperone gp96, an endoplasmic reticulum paralog of HSP90, in fact blocks HSP90-LPS interaction. It inhibited the binding of LPS to the cell surface of both wild type and gp96-null cells and thereby abrogated the cellular response to LPS but not to other Toll-like receptor ligands. We also generated a series of peptide derivatives (named peptide inhibitors of endotoxin responsiveness (PIERs)) from the N-terminal helix structure of HSP90 and demonstrated their effectiveness in blocking LPS activity. PIER inhibition of LPS signaling was partially reversed by CD14 expression. Moreover, we found that a cell-permeable PIER abrogated HSP90 function and caused degradation of multiple known HSP90 client proteins in cancer cells. Thus, targeting HSP90 is a promising modality for treatment of both LPS-mediated pathology and cancer.

Thalidomide inhibits lipopolysaccharide-induced nitric oxide production and prevents lipopolysaccharide-mediated lethality in mice.

The effect of thalidomide on lipopolysaccharide-induced nitric oxide (NO) production was studied using RAW 264.7 macrophage-like cells. Thalidomide significantly inhibited lipopolysaccharide-induced NO production via reduced expression of an inducible NO synthase. Thalidomide reduced the phosphorylation of the p65 nuclear factor-kappaB subunit, inhibitory kappaB (IkappaB) and IkappaB kinase in lipopolysaccharide-stimulated cells. However, thalidomide did not affect the expression of interferon-beta (IFN-beta) and interferon regulatory factor-1 in response to lipopolysaccharide. Further, thalidomide inhibited the MyD88 augmentation in lipopolysaccharide-stimulated cells, whereas it did not alter the expression of TIR domain-containing adaptor-inducing IFN-beta in the MyD88-independent pathway. Thalidomide significantly inhibited the NO production in response to Pam(3)Cys, CpG DNA and imiquimod as MyD88-dependent Toll-like receptor (TLR) ligands, but not polyI:C as a MyD88-independent TLR ligand. Therefore, thalidomide was suggested to inhibit lipopolysaccharide-induced NO production via downregulation of the MyD88-dependent signal pathway. The anti-inflammatory action of thalidomide might be involved in the prevention of lipopolysaccharide-mediated lethality in mice.

Thalidomide inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production via down-regulation of MyD88 expression.

The effect of thalidomide on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was studied by using RAW 264.7 murine macrophage-like cells. Thalidomide significantly inhibited LPS-induced TNF-alpha production. Thalidomide prevented the activation of nuclear factor (NF)-KB by down-regulating phosphorylation of inhibitory KB factor (IKB), and IKB kinase (IKK)-alpha and IKK-beta Moreover, thalidomide inhibited LPS-induced phosphorylation of AKT, p38 and stress-activated protein kinase (SAPK)/JNK. The expression of myeloid differentiation factor 88 (MyD88) protein and mRNA was markedly reduced in thalidomide-treated RAW 264.7 cells but there was no significant alteration in the expression of interleukin-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6 in the cells. Thalidomide did not affect the cell surface expression of Toll-like receptor (TLR) 4 and CD14, suggesting the impairment of intracellular LPS signalling in thalidomide-treated RAW 264.7 cells. Thalidomide significantly inhibited the TNF-alpha production in response to palmitoyl-Cys(RS)-2,3-di(palmitoyloxy) propyl)-Ala-Gly-OH (Pam(3)Cys) as a MyD88-dependent TLR2 ligand. Therefore, it is suggested that thalidomide might impair LPS signalling via down-regulation of MyD88 protein and mRNA and inhibit LPS-induced TNF-alpha production. The putative mechanism of thalidomide-induced MyD88 down-regulation is discussed.

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation.

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.