Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Applying a Gene Reversal Rate Computational Methodology to Identify Drugs for a Rare Cancer: Inflammatory Breast Cancer.

The aim of this study was to utilize a computational methodology based on Gene Reversal Rate (GRR) scoring to repurpose existing drugs for a rare and understudied cancer: inflammatory breast cancer (IBC). This method uses IBC-related gene expression signatures (GES) and drug-induced gene expression profiles from the LINCS database to calculate a GRR score for each candidate drug, and is based on the idea that a compound that can counteract gene expression changes of a disease may have potential therapeutic applications for that disease. Genes related to IBC with associated differential expression data (265 up-regulated and 122 down-regulated) were collated from PubMed-indexed publications. Drug-induced gene expression profiles were downloaded from the LINCS database and candidate drugs to treat IBC were predicted using their GRR scores. Thirty-two (32) drug perturbations that could potentially reverse the pre-compiled list of 297 IBC genes were obtained using the LINCS Canvas Browser (LCB) analysis. Binary combinations of the 32 perturbations were assessed computationally to identify combined perturbations with the highest GRR scores, and resulted in 131 combinations with GRR greater than 80%, that reverse up to 264 of the 297 genes in the IBC-GES. The top 35 combinations involve 20 unique individual drug perturbations, and 19 potential drug candidates. A comprehensive literature search confirmed 17 of the 19 known drugs as having either anti-cancer or anti-inflammatory activities. AZD-7545, BMS-754807, and nimesulide target known IBC relevant genes: PDK, Met, and COX, respectively. AG-14361, butalbital, and clobenpropit are known to be functionally relevant in DNA damage, cell cycle, and apoptosis, respectively. These findings support the use of the GRR approach to identify drug candidates and potential combination therapies that could be used to treat rare diseases such as IBC.

Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia.

OBJECTIVE: NRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DESIGN: We performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. RESULTS: Using high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. CONCLUSION: We have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic for NRF2high ESCC.

Obesity and Breast Cancer Metastasis across Genomic Subtypes.

BACKGROUND: Obese women have higher risk of aggressive breast tumors and distant metastasis. However, obesity has rarely been assessed in association with metastasis in diverse populations. METHODS: In the Carolina Breast Cancer Study Phase 3 (2008-2013), waist-to-hip ratio (WHR), body mass index (BMI), and molecular subtype [PAM50 risk-of-recurrence (ROR) score] were assessed. Obesity measures were evaluated in association with metastasis within five years of diagnosis, overall and stratified by race and ROR score. Absolute risk of metastasis and risk differences between strata were calculated using the Kaplan-Meier estimator, adjusted for age, grade, stage, race, and ER status. Relative frequency of metastatic site and multiplicity were estimated in association with obesity using generalized linear models. RESULTS: High-WHR was associated with higher risk of metastasis (5-year risk difference, RD, 4.3%; 95% confidence interval, 2.2-6.5). It was also associated with multiple metastases and metastases at all sites except brain. The 5-year risk of metastasis differed by race (11.2% and 6.9% in Black and non-Black, respectively) and ROR score (19.5% vs. 6.6% in high vs. low-to-intermediate ROR-PT). Non-Black women and those with low-to-intermediate ROR scores had similar risk in high- and low-WHR strata. However, among Black women and those with high ROR, risk of metastasis was elevated among high-WHR (RDBlack/non-Black = 4.6%, RDHigh/Low-Int = 3.1%). Patterns of metastasis were similar by BMI. CONCLUSIONS: WHR is associated with metastatic risk, particularly among Black women and those with high-risk tumors. IMPACT: Understanding how risk factors for metastasis interact may help in tailoring care plans and surveillance among patients with breast cancer.

Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences.

Aggressive breast cancer variants, like triple negative and inflammatory breast cancer, contribute to disparities in survival and clinical outcomes among African American (AA) patients compared to White (W) patients. We previously identified the dominant role of anti-apoptotic protein XIAP in regulating tumor cell adaptive stress response (ASR) that promotes a hyperproliferative, drug resistant phenotype. Using The Cancer Genome Atlas (TCGA), we identified 46-88 ASR genes that are differentially expressed (2-fold-change and adjusted p-value < 0.05) depending on PAM50 breast cancer subtype. On average, 20% of all 226 ASR genes exhibited race-related differential expression. These genes were functionally relevant in cell cycle, DNA damage response, signal transduction, and regulation of cell death-related processes. Moreover, 23% of the differentially expressed ASR genes were associated with AA and/or W breast cancer patient survival. These identified genes represent potential therapeutic targets to improve breast cancer outcomes and mitigate associated health disparities.

Racial differences in breast cancer outcomes by hepatocyte growth factor pathway expression.

PURPOSE: Black women have a 40% increased risk of breast cancer-related mortality. These outcome disparities may reflect differences in tumor pathways and a lack of targetable therapies for specific subtypes that are more common in Black women. Hepatocyte growth factor (HGF) is a targetable pathway that promotes breast cancer tumorigenesis, is associated with basal-like breast cancer, and is differentially expressed by race. This study assessed whether a 38-gene HGF expression signature is associated with recurrence and survival in Black and non-Black women. METHODS: Study participants included 1957 invasive breast cancer cases from the Carolina Breast Cancer Study. The HGF signature was evaluated in association with recurrence (n = 1251, 171 recurrences), overall, and breast cancer-specific mortality (n = 706, 190/328 breast cancer/overall deaths) using Cox proportional hazard models. RESULTS: Women with HGF-positive tumors had higher recurrence rates [HR 1.88, 95% CI (1.19, 2.98)], breast cancer-specific mortality [HR 1.90, 95% CI (1.26, 2.85)], and overall mortality [HR 1.69; 95% CI (1.17, 2.43)]. Among Black women, HGF positivity was significantly associated with higher 5-year rate of recurrence [HR 1.73; 95% CI (1.01, 2.99)], but this association was not significant in non-Black women [HR 1.68; 95% CI (0.72, 3.90)]. Among Black women, HGF-positive tumors had elevated breast cancer-specific mortality [HR 1.80, 95% CI (1.05, 3.09)], which was not significant in non-Black women [HR 1.52; 95% CI (0.78, 2.99)]. CONCLUSION: This multi-gene HGF signature is a poor-prognosis feature for breast cancer and may identify patients who could benefit from HGF-targeted treatments, an unmet need for Black and triple-negative patients.

Pharmacological activation of the Sonic hedgehog pathway with a Smoothened small molecule agonist ameliorates the severity of alcohol-induced morphological and behavioral birth defects in a zebrafish model of fetal alcohol spectrum disorder.

Ethanol exposure during the early stages of embryonic development can lead to a range of morphological and behavioral differences termed fetal alcohol spectrum disorders (FASDs). In a zebrafish model, we have shown that acute ethanol exposure at 8-10 hr postfertilization (hpf), a critical time of development, produces birth defects similar to those clinically characterized in FASD. Dysregulation of the Sonic hedgehog (Shh) pathway has been implicated as a molecular basis for many of the birth defects caused by prenatal alcohol exposure. We observed in zebrafish embryos that shh expression was significantly decreased by ethanol exposure at 8-10 hpf, while smo expression was much less affected. Treatment of zebrafish embryos with SAG or purmorphamine, small molecule Smoothened agonists that activate Shh signaling, ameliorated the severity of ethanol-induced developmental malformations including altered eye size and midline brain development. Furthermore, this rescue effect of Smo activation was dose dependent and occurred primarily when treatment was given after ethanol exposure. Markers of Shh signaling (gli1/2) and eye development (pax6a) were restored in embryos treated with SAG post-ethanol exposure. Since embryonic ethanol exposure has been shown to produce later-life neurobehavioral impairments, juvenile zebrafish were examined in the novel tank diving test. Our results further demonstrated that in zebrafish embryos exposed to ethanol, SAG treatment was able to mitigate long-term neurodevelopmental impairments related to anxiety and risk-taking behavior. Our results indicate that pharmacological activation of the Shh pathway at specific developmental timing markedly diminishes the severity of alcohol-induced birth defects.

Optimization and validation of a DYRK1A TR-FRET assay for high-throughput screening.

Due to its role in brain development, the DYRK1A kinase (dual-specificity tyrosine phosphorylation-regulated kinase 1a) has been proposed as a drug target for Down syndrome, and diseases associated with neurodegeneration including Alzheimer's and Parkinson's. Other diseases in which DYRK1A is implicated include cancer and diabetes. Hence, there is need for potent and selective DYRK1A inhibitors. To screen large diversity compound libraries versus DYRK1A requires the development of a cost-effective high-throughput screen. In this study, we have taken a commercial time-resolved fluorescence energy transfer (TR-FRET)-based assay for DYRK1A and optimized for smaller volumes and homogenous format at room temperature. Tracer and enzyme concentrations were determined. DYRK1A-GST, anti-GST Ab and tracer were pre-combined and total assay volume reduced 2-fold. The assay was validated using whole plate minimum and maximum signal wells with a Z' of 0.7-0.8 determined. Overall, this method:•Results in an optimized low volume, homogenous and validated assay for DYRK1A.•Delivers a cost effective high-throughput assay format for DYRK1A inhibitor screening.

Development and validation of a hedgehog heparin-binding assay for high-throughput screening.

Sonic hedgehog (Shh) is a morphogenic protein with critical roles in embryogenesis and the development of some cancers. Hence, identifying inhibitors of the Shh pathway is of great therapeutic value. Heparin and HSPGs act as crucial modulators of Shh activity. To identify molecules that antagonize Shh binding to heparin we have developed a solid-phase plate-based assay. The N-terminal domain of Shh (ShhN) protein is first coated in 384-well plates and the direct binding of fluorescein-labeled heparin (flu-heparin) assessed by measuring the fluorescence signal after incubation and wash steps. Binding of ShhN protein to the 384-well plates was confirmed and optimized by a standard ELISA using a monoclonal antibody recognizing folded ShhN. The assay was validated using whole plate minimum and maximum signal wells with a Z' of 0.68-0.75 determined. Herein, we describe the development and validation of a high throughput screen to identify small molecule antagonists of Shh heparin binding. Overall, this method•Results in an optimized and validated assay for hedgehog heparin binding.•Delivers a cost effective high-throughput screen format for hedgehog heparin antagonist screening.

Data supporting a pilot high-throughput screen of a drug library for identification of DYRK1A inhibitors and high-content imaging analysis of identified harmine analogs.

The data presented in this article support the accompanying research article "Identification of harmine and ß-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies" [1]. As DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) plays a role in the pathophysiology of a number of diseases including diabetes, cancer and neurodegeneration [2], [3], [4], the identification of DYRK1A inhibitors is of significant interest. This data article details the hits identified from a DYRK1A high-throughput screen of a small molecule compound library containing over 95% approved drugs. Twenty-two compounds were identified with >50% inhibition, including harmine and four of its analogs. Subsequent profiling of these harmine analogs using glioma cancer cell lines and high-content image analysis identified those with effects on growth and cytotoxicity.

Identification of harmine and ß-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies.

DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the ß-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM) cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.

Literature-Wide Association Studies (LWAS) for a Rare Disease: Drug Repurposing for Inflammatory Breast Cancer.

Drug repurposing is an effective means for rapid drug discovery. The aim of this study was to develop and validate a computational methodology based on Literature-Wide Association Studies (LWAS) of PubMed to repurpose existing drugs for a rare inflammatory breast cancer (IBC). We have developed a methodology that conducted LWAS based on the text mining technology Word2Vec. 3.80 million "cancer"-related PubMed abstracts were processed as the corpus for Word2Vec to derive vector representation of biological concepts. These vectors for drugs and diseases served as the foundation for creating similarity maps of drugs and diseases, respectively, which were then employed to find potential therapy for IBC. Three hundred and thirty-six (336) known drugs and three hundred and seventy (370) diseases were expressed as vectors in this study. Nine hundred and seventy (970) previously known drug-disease association pairs among these drugs and diseases were used as the reference set. Based on the hypothesis that similar drugs can be used against similar diseases, we have identified 18 diseases similar to IBC, with 24 corresponding known drugs proposed to be the repurposing therapy for IBC. The literature search confirmed most known drugs tested for IBC, with four of them being novel candidates. We conclude that LWAS based on the Word2Vec technology is a novel approach to drug repurposing especially useful for rare diseases.

Identification of a Triple Drug Combination That Is Synergistically Cytotoxic for Triple-Negative Breast Cancer Cells Using a Novel Combination Discovery Approach.

Triple-negative breast cancer (TNBC) is a very aggressive form of breast cancer with few molecularly targeted therapies. We used a novel unbiased approach to identify higher-order synergistic or enhancer combinations of marketed kinase inhibitor drugs that inhibit cell viability of TNBC cell lines. We mixed all 33 kinase-targeted drugs on the market at the time of this study, which allowed for all possible combinations to exist in the initial mixture. A kinase inhibitor group dropout approach was used to identify active groups and then single active drugs. After only three rounds of deconvolution, we identified five single drugs to test further. After further testing, we focused on one novel subset consisting of three kinase inhibitor drugs: dasatinib, afatinib, and trametinib (DAT) that target src family kinases, HER2/EGFR, and MEK, respectively. The DAT combination potently inhibited the proliferation of three TNBC cell lines and modestly inhibited a fourth. However, it was not significantly more potent or synergistic than other two drug combinations of these drugs. The cytotoxic activities of all possible combinations of these three drugs were also analyzed. Compared with all two-way combinations, the three-way DAT combination generated the most cytotoxicity and the highest synergies for two of the four cell lines tested, with possibly mild synergy in a third cell line. These data indicated that the DAT combination should be evaluated for efficacy in an in vivo model of TNBC and may provide a novel combination of existing drugs for the treatment of a subset of TNBC cases.

Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism.

BACKGROUND: Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development. METHODS: Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior. RESULTS: Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids. CONCLUSIONS: These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.

An innovative educational program for addressing health disparities in translational cancer research.

North Carolina Central University (NCCU) and Duke Cancer Institute implemented an NCI-funded Translational Cancer Disparities Research Partnership to enhance translational cancer research, increase the pool of underrepresented racial and ethnic group (UREG) researchers in the translational and clinical research workforce, and equip UREG trainees with skills to increase diversity in clinical trials. The Cancer Research Education Program (C-REP) provided training for UREG graduate students and postdoctoral fellows at Duke and NCCU. An innovative component of C-REP is the Translational Immersion Experience (TIE), which enabled Scholars to gain knowledge across eight domains of clinical and translational research (clinical trials operations, data monitoring, regulatory affairs, UREG accrual, biobanking, community engagement, community outreach, and high-throughput drug screening). Program-specific evaluative metrics were created for three broad domains (clinical operations, basic science/lab research, and population-based science) and eight TIE domains. Two cohorts (n = 13) completed pre- and post-surveys to determine program impact and identify recommendations for program improvement. Scholars reported statistically significant gains in knowledge across three broad domains of biomedical research and seven distinct areas within TIE. Training in translational research incorporating immersions in clinical trials operation, biobanking, drug development, and community engagement adds value to career development of UREG researchers.

Cannabinoids Exacerbate Alcohol Teratogenesis by a CB1-Hedgehog Interaction.

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ9-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.

Perspectives on Inflammatory Breast Cancer (IBC) Research, Clinical Management and Community Engagement from the Duke IBC Consortium.

Inflammatory breast cancer (IBC) is an understudied and aggressive form of breast cancer with a poor prognosis, accounting for 2-6% of new breast cancer diagnoses but 10% of all breast cancer-related deaths in the United States. Currently there are no therapeutic regimens developed specifically for IBC, and it is critical to recognize that all aspects of treating IBC - including staging, diagnosis, and therapy - are vastly different than other breast cancers. In December 2014, under the umbrella of an interdisciplinary initiative supported by the Duke School of Medicine, researchers, clinicians, research administrators, and patient advocates formed the Duke Consortium for IBC to address the needs of patients in North Carolina (an ethnically and economically diverse state with 100 counties) and across the Southeastern United States. The primary goal of this group is to translate research into action and improve both awareness and patient care through collaborations with local, national and international IBC programs. The consortium held its inaugural meeting on Feb 28, 2018, which also marked Rare Disease Day and convened national research experts, clinicians, patients, advocates, government representatives, foundation leaders, staff, and trainees. The meeting focused on new developments and challenges in the clinical management of IBC, research challenges and opportunities, and an interactive session to garner input from patients, advocates, and community partners that would inform a strategic plan toward continuing improvements in IBC patient care, research, and education.

Lapatinib-induced annexin A6 upregulation as an adaptive response of triple-negative breast cancer cells to EGFR tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.

Targeted therapy of esophageal squamous cell carcinoma: the NRF2 signaling pathway as target.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease that requires extensive research. Here, we review the current understanding of the functions of the nuclear factor erythroid-derived 2-like 2 (NRF2) signaling pathway in the esophagus. Genomic data suggest that gene mutations and several other mechanisms result in NRF2 hyperactivation in human ESCC. As a consequence, NRF2high ESCC is more resistant to chemoradiotherapy and associated with poorer survival than NRF2low ESCC. Mechanistically, we believe NRF2, functioning as a transcription factor, causes an esophageal phenotype through regulation of gene transcription. We discuss metabolism, mitochondria, proteasomes, and several signaling pathways as downstream players that may contribute to an esophageal phenotype due to NRF2 hyperactivation. Finally, strategies are proposed to target the NRF2 signaling pathway for therapy of NRF2high ESCC.

Data on peptides identified by mass spectrometry analysis of in vitro DYRK1A-mediated phosphorylation sites on GLI1.

The data presented in this article support the accompanying research article "Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1" (Ehe et al., 2017) [1]. Although it has been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate the hedgehog pathway transcription factor GLI1 (GLIoma-associated oncogene homolog 1) and promote its nuclear localization, the DYRK1A-mediated sites of phosphorylation on GLI1 involved were not fully known. This article details the mass spectrometry methods and resulting dataset for the peptides identified from GLI1 when incubated with DYRK1A under varying conditions. The data include details of sequence coverage and all phospho-peptides identified.