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

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

The DEK Oncogene Is a Target of Steroid Hormone Receptor Signaling in Breast Cancer

<div><p>Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated <em>in vitro</em> following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify <em>DEK</em> as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.</p> </div

RNAseq data.

(XLSX)</p

GADD34 overexpression during prolonged lapatinib treatment allows to overcome ER stress response-mediated inhibition of protein synthesis.

A) Protein levels of GADD34 (PPP1R15A) in the indicated conditions. B-C) Western blot of p-eIF2 (S51) under indicated conditions with and without treatment with guanabenz (GADD34 inhibitor) (B) or GADD34-targeting shRNA (C). D-E) Relative viability of cells at the control (parental) and relapse (2M) stages in response to 72hr treatment with guanabenz (D) or shRNA against GADD34 (E). F-G) Protein synthesis rate measurement with Click-iT Protein synthesis kit in the indicated stages of lapatinib treatment with and without guanabenz (F) or shRNA targeting GADD34 (G). Data is presented as mean fluorescence intensity (MFI). H) A model summarizing the signaling, metabolic and proteostatic changes during the acquisition of resistance to lapatinib in SKBR3 cells. I) mRNA levels of PPP1R15A in the cohort of advanced Her2+ breast cancer patients (n = 53 samples) with short (3 years) duration of response to first-line trastuzumab therapy, from the Long-HER study (33). Statistics: Error bars show standard deviations of 2 (D-E) or 3 (F-G) replicate samples. Data are representative of at least 2 independent experiments (D,F). ***: P S1 Table.</p

The GADD34-eIF2 axis plays an important role in Her3-mediated growth during chronic Her2 inhibition in BT474 cells.

(PDF)</p

Expression profiles of genes with the most overexpression in the relapsed samples.

A) heatmap of top genes whose expression was increased in the relapse (2M) samples (from RNAseq). B) Bar plot of expression values for PPP1R15A (from RNAseq) in the indicated samples. (PDF)</p

DEK is necessary for 17β-estradiol stimulated cell proliferation and modulates sensitivity to tamoxifen.

<p>(A) DEK expression is required for 17β-estradiol stimulated cellular proliferation. Hormone starved MCF7 cells transduced with non-targeting shRNA (NTsh) or DEK shRNA (DEKsh2) were untreated (CS-FBS) or exposed to 10 nM 17β-estradiol, then cultured in BrdU. The percentage of BrdU positive cells was determined by flow cytometry. Asterisk (*) denotes p<0.05 using Student’s t-test. (B and C) DEK depletion by shRNA (DEKsh2) works synergistically with tamoxifen to induce apoptosis in breast cancer cell lines. (B) Bright field images (100× magnification) of MCF7 cells expressing either NTsh or DEKsh2 were cultured in low serum media and either untreated or treated with tamoxifen for 18 hours. (C) DEK depletion by shRNA (DEKsh2) enhances the cytotoxic effect of tamoxifen. DEK proficient and deficient MCF7 (left) and T47D (right) cells were grown in low serum media then treated with 3 µg/ml tamoxifen for 22 hours. Cells were labeled with 7AAD then analyzed for sub-G1 content by flow cytometry as a measure of apoptosis. Results shown are the average of triplicate experiments. Two asterisks (**) indicate p<0.01 as determined using a 2-way ANOVA test for significance. For MCF7 cells, p = 0.08. (A and B insets) DEK shRNA knockdown is shown by western blot analysis for normally cultured cells that were transduced with lentivirus carrying either non-targeting shRNA (NTsh) or DEK specific shRNA (DEKsh2).</p

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