Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. EXPERIMENTAL DESIGN: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. RESULTS: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. CONCLUSIONS: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes

Human Papillomavirus Oral- and Sero- Positivity in Fanconi Anemia

High-risk human papillomavirus (HPV) is prevalent and known to cause 5% of all cancers worldwide. The rare, cancer prone Fanconi anemia (FA) population is characterized by a predisposition to both head and neck squamous cell carcinomas and gynecological cancers, but the role of HPV in these cancers remains unclear. Prompted by a patient-family advocacy organization, oral HPV and HPV serological studies were simultaneously undertaken. Oral DNA samples from 201 individuals with FA, 303 unaffected family members, and 107 unrelated controls were tested for 37 HPV types. Serum samples from 115 individuals with FA and 55 unrelated controls were tested for antibodies against 9 HPV types. Oral HPV prevalence was higher for individuals with FA (20%) versus their parents (13%; p = 0.07), siblings (8%, p = 0.01), and unrelated controls (6%, p ≤ 0.001). A FA diagnosis increased HPV positivity 4.84-fold (95% CI: 1.96-11.93) in adjusted models compared to unrelated controls. Common risk factors associated with HPV in the general population did not predict oral positivity in FA, unlike unrelated controls. Seropositivity and anti-HPV titers did not significantly differ in FA versus unrelated controls regardless of HPV vaccination status. We conclude that individuals with FA are uniquely susceptible to oral HPV independent of conventional risk factors

Drug resistance mechanisms create targetable proteostatic vulnerabilities in Her2+ breast cancers

Oncogenic kinase inhibitors show short-lived responses in the clinic due to high rate of acquired resistance. We previously showed that pharmacologically exploiting oncogene-induced proteotoxic stress can be a viable alternative to oncogene-targeted therapy. Here, we performed extensive analyses of the transcriptomic, metabolomic and proteostatic perturbations during the course of treatment of Her2+ breast cancer cells with a Her2 inhibitor covering the drug response, resistance, relapse and drug withdrawal phases. We found that acute Her2 inhibition, in addition to blocking mitogenic signaling, leads to significant decline in the glucose uptake, and shutdown of glycolysis and of global protein synthesis. During prolonged therapy, compensatory overexpression of Her3 allows for the reactivation of mitogenic signaling pathways, but fails to re-engage the glucose uptake and glycolysis, resulting in proteotoxic ER stress, which maintains the protein synthesis block and growth inhibition. Her3-mediated cell proliferation under ER stress during prolonged Her2 inhibition is enabled due to the overexpression of the eIF2 phosphatase GADD34, which uncouples protein synthesis block from the ER stress response to allow for active cell growth. We show that this imbalance in the mitogenic and proteostatic signaling created during the acquired resistance to anti-Her2 therapy imposes a specific vulnerability to the inhibition of the endoplasmic reticulum quality control machinery. The latter is more pronounced in the drug withdrawal phase, where the de-inhibition of Her2 creates an acute surge in the downstream signaling pathways and exacerbates the proteostatic imbalance. Therefore, the acquired resistance mechanisms to oncogenic kinase inhibitors may create secondary vulnerabilities that could be exploited in the clinic

Metabolomic reprogramming during chronic lapatinib treatment.

A) Heatmap of the most notable altered intracellular metabolites between the different stages of 1μM lapatinib treatment, measured by NMR mass spectrometry. B) A model summarizing the effect of chronic lapatinib treatment on the glucose, energy and amino acid metabolism. Blue lines: reduced flux, red lines: increased flux at the relapse stage (2M). C) Western blot of O-GlcNAc-conjugated protein levels in the indicated conditions. O-GlcNAc levels may serve as a readout of the N-linked hexosamine (GlcNAc) levels. D) Intracellular glucose uptake assay measuring 2-deoxyglucose uptake with and without insulin after lapatinib treatment in SKBR3 cells. Her3 overexpression is unable to rescue the glucose uptake inhibition of lapatinib treatment. E) Overexpression of Her2 or Her3 in the non-transformed MCF10A cells induces similar activation of downstream pathways (p-Akt). F) Glucose uptake rates with control, Her2 or Her3 overexpression in MCF10A cells. Statistics: error bars show standard deviations from 2 replicate samples. Data are representative of 2 independent experiments. ***: P S1 Table.</p

RNAseq data.

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Therapeutic reduction of lysophospholipids in the digestive tract recapitulates the metabolic benefits of bariatric surgery and promotes diabetes remission

Objective: Obesity and obesity-related metabolic disorders are major health problems worldwide. The most effective obesity intervention is bariatric surgery. This study tested the hypothesis that bariatric surgery alters phospholipid metabolism in the gastrointestinal tract to favor a metabolically healthy gut microbiota profile and therapeutic intervention of phospholipid metabolism in the gastrointestinal may have similar metabolic benefits. Methods: The first study compared plasma levels of the bioactive lipid metabolites lysophospholipid and trimethylamine N-oxide (TMAO) as well as gut microbiota profile in high fat/carbohydrate (HFHC) diet-fed C57BL/6 mice with or without vertical sleeve gastrectomy (VSG) and in Pla2g1b−/− mice with group 1B phospholipase A2 gene inactivation. The second study examined the effectiveness of the non-absorbable secretory phospholipase A2 inhibitor methyl indoxam to reverse hyperglycemia and hyperlipidemia in HFHC diet-fed C57BL/6 mice after diabetes onset. Results: Both bariatric surgery and PLA2G1B inactivation were shown to reduce lysophospholipid content in the gastrointestinal tract, resulting in resistance to HFHC diet-induced alterations of the gut microbiota, reduction of the cardiovascular risk factors hyperlipidemia and TMAO, decreased adiposity, and prevention of HFHC diet-induced diabetes. Importantly, treatment of wild type mice with methyl indoxam after HFHC diet-induced onset of hyperlipidemia and hyperglycemia effectively restored normal plasma lipid and glucose levels and replicated the metabolic benefits of VSG surgery with diabetes remission and TMAO reduction. Conclusion: These results provided pre-clinical evidence that PLA2G1B inhibition in the digestive tract may be a viable alternative option to bariatric surgery for obesity and obesity-related cardiometabolic disorder intervention. Keywords: Phospholipase A2, Gut microbiota, Cardiometabolic disease, Bariatric surgery, Lysophospholipi

Her3 overexpression confers tumor cell growth under lapatinib.

A) Total and phospho-Her3 levels at the indicated stages of lapatinib (1μM) treatment. B) Western blot of the indicated signaling proteins with and without shRNA-mediated knock-down of Her3 at the indicated stages of lapatinib treatment. C) Relative viability (compared to control) under the same conditions. D) Her3 was stably silenced or overexpressed in SKBR3 cells, and E) the relative cell growth (relative to control) was calculated after 1μM lapatinib treatment for the indicated time period. Statistics: error bars show standard deviations of 6 (C) or 3 (E) replicate experiments. ***: P S1 Table.</p

Protein homeostasis dynamics during the course of chronic lapatinib treatment.

A) Immunofluorescence images of at the indicated stages of 1μM lapatinib treatment stained for newly synthesized proteins within a 30 min window using the Click-iT Protein Synthesis assay. B) Flow cytometry-based measurement of protein synthesis using the same conditions as in A using the Click-iT assay. Data is presented as mean fluorescence intensity (MFI). C) Protein synthesis rate measurement as in (B) in the indicated conditions with and without Her3 silencing. D) Western blots of the indicated ER stress response markers in the indicated conditions. Statistics: error bars show standard deviations of 2 (B-C) replicates. The data are representative of at least 2 independent experiments. ***: P S1 Table.</p