New insights into redox-related risk factors and therapeutic targets in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common cancer in the oral cavity accounting for 90 % of oral cancer with a global incidence of 350.000 new cases per year. Curative resection along with adjuvant radiation therapy or a combination of radiotherapy with chemotherapy remain as gold standard in treating OSCC. Still, local recurrence, lymph nodal recurrence, and complications of radiation remain the main cause of tumor-related mortality. Reactive oxygen species are not only correlated to the etiology of OSCC due to oxidative DNA damage, lipid peroxidation or effecting signal transduction cascades that effect cell proliferation and tumorigenesis, but are also of great interest in the therapy of OSCC patients. As induced oxidative stress can be used therapeutically for the induction of tumor cell death, redox targets might be a therapeutic addition to the conventional treatment options. In this review, we discuss markers of impaired redox homeostasis as well as potential redox-related treatment targets in OSCC.

NTRK fusion events and targeted treatment of advanced radioiodine refractory thyroid cancer.

PURPOSE: Pathogenic fusion events involving neurotrophic receptor tyrosine kinase (NTRK) have been described in ~ 2% of differentiated thyroid cancer (DTC). The selective tropomyosin receptor kinase (TRK) inhibitors entrectinib and larotrectinib have been approved in a tumor agnostic manner based on phase 1/2 clinical trials. In a real-world setting at five referral centers, we aimed to describe the prevalence of NTRK gene fusions and the efficacy and safety of TRK inhibitor treatment for non-medullary, advanced thyroid cancer (TC). METHODS: A total of 184 TC patients with testing for NTRK gene fusions were included. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan-Meier method in six patients with NTRK fusion-positive TC who underwent TRK inhibitor therapy. RESULTS: 8/184 (4%) patients harbored NTRK gene fusions. Six patients with radioiodine (RAI)-refractory TC harboring NTRK1 (n = 4) and NTRK3 (n = 2) gene fusions were treated with larotrectinib. Five patients (83%) had received ≥ 1 prior systemic therapy and one patient did not receive prior systemic therapy. All patients had morphologically progressive disease before treatment initiation. Objective response rate was 83%, including two complete remissions. Median PFS from start of TRK inhibitor treatment was 23 months (95% confidence interval [CI], 0-57.4) and median OS was not reached (NR) (95% CI, NR). Adverse events were of grade 1-3. CONCLUSION: The prevalence of NTRK gene fusions in our cohort of RAI-refractory TC is slightly higher than reported for all TC patients. Larotrectinib is an effective treatment option in the majority of NTRK gene fusion-positive advanced TC patients after prior systemic treatment and has a favorable safety profile.

Implementation of Precision Oncology for Patients with Metastatic Breast Cancer in an Interdisciplinary MTB Setting.

The advent of molecular diagnostics and the rising number of targeted therapies have facilitated development of precision oncology for cancer patients. In order to demonstrate its impact for patients with metastatic breast cancer (mBC), we initiated a Molecular Tumor Board (MTB) to provide treatment recommendations for mBC patients who had disease progression under standard treatment. NGS (next generation sequencing) was carried out using the Oncomine multi-gene panel testing system (Ion Torrent). The MTB reviewed molecular diagnostics' results, relevant tumor characteristics, patient's course of disease and made personalized treatment and/or diagnostic recommendations for each patient. From May 2017 to December 2019, 100 mBC patients were discussed by the local MTB. A total 72% of the mBC tumors had at least one molecular alteration (median 2 per case, range: 1 to 6). The most frequent genetic changes were found in the following genes: PIK3CA (19%) and TP53 (17%). The MTB rated 53% of these alterations as actionable and treatment recommendations were made accordingly for 49 (49%) patients. Sixteen patients (16%) underwent the suggested therapy. Nine out of sixteen patients (56%; 9% of all) experienced a clinical benefit with a progression-free survival ratio ≥ 1.3. Personalized targeted therapy recommendations resulting from MTB case discussions could provide substantial benefits for patients with mBC and should be implemented for all suitable patients.

NGS-guided precision oncology in metastatic breast and gynecological cancer: first experiences at the CCC Munich LMU.

PURPOSE: Comprehensive genomic profiling identifying actionable molecular alterations aims to enable personalized treatment for cancer patients. The purpose of this analysis was to retrospectively assess the impact of personalized recommendations made by a multidisciplinary tumor board (MTB) on the outcome of patients with breast or gynecological cancers, who had progressed under standard treatment. Here, first experiences of our Comprehensive Cancer Center Molecular Tumor Board are reported. METHODS: All patients were part of a prospective local registry. 95 patients diagnosed with metastatic breast cancer or gynecological malignancies underwent extended molecular profiling. From May 2017 through March 2019, the MTB reviewed all clinical cases considering tumor profile and evaluated molecular alterations regarding further diagnostic and therapeutic recommendations. RESULTS: 95 patients with metastatic breast or gynecological cancers were discussed in the MTB (68% breast cancer, 20% ovarian cancer, 5% cervical cancer, 3% endometrial cancer and 4% others). Genes with highest mutation rate were PIK3CA and ERBB2. Overall, 34 patients (36%) received a biomarker-based targeted therapy recommendation. Therapeutic recommendations were implemented in nine cases; four patients experienced clinical benefit with a partial response or disease stabilization lasting over 4 months. CONCLUSION: In the setting of a multidisciplinary molecular tumor board, a small but clinically meaningful group of breast and gynecological cancer patients benefits from comprehensive genomic profiling. Broad and successful implementation of precision medicine is complicated by patient referral at late stage disease and limited access to targeted agents and early clinical trials. TRIAL REGISTRATION NUMBER: 284-10 (03.05.2018).

POLE gene hotspot mutations in advanced pancreatic cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, lacking relevant prognostic and predictive biomarkers. DNA polymerase epsilon (POLE) has important functions in the maintenance of genetic stability during DNA replication and has previously been associated with favorable prognosis in endometrial and colorectal cancer. However, its relevance in advanced pancreatic cancer (aPDAC) has not been examined to date. METHODS: Using pyrosequencing on tumoral DNA extracted from 60 samples from the AIO-PK0104 study as well as 55 samples from completed translational trials, we examined POLE hotspot mutations in exon 9 (P286R) and exon 13 (V411R/L/M) in the POLE gene exonuclease domain. DNA extracted from 37 endometrial carcinomas were tested as positive controls. Publically available sequencing databases were searched for POLE mutations in PDAC samples. RESULTS: Fifty-three patients (pts) were men, 62 pts were women, median age was 61.2 years. Median overall survival (OS) was 7.4 months and median progression free survival (PFS) was 4.0 months. In four of the 37 endometrial carcinomas POLE mutations were detected in exon 9 (10.8%) and none in exon 13. In none of the overall 115 aPDAC tumors POLE gene hotspot mutations could be detected. CONCLUSION: Mutations in the hotspot regions of exon 9 and 13 of the POLE gene are very rare events in advanced pancreatic cancer. Thus, it is unlikely that POLE gene mutations contribute to genetic instability in the vast majority of aPDAC. POLE mutation does not serve as a relevant biomarker and should not be tested on a regular basis in PDAC.

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo.

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3).

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities.

Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies.

Insulin inhibits lipolysis in adipocytes via the evolutionarily conserved mTORC1-Egr1-ATGL-mediated pathway.

One of the basic functions of insulin in the body is to inhibit lipolysis in adipocytes. Recently, we have found that insulin inhibits lipolysis and promotes triglyceride storage by decreasing transcription of adipose triglyceride lipase via the mTORC1-mediated pathway (P. Chakrabarti et al., Diabetes 59:775-781, 2010), although the mechanism of this effect remained unknown. Here, we used a genetic screen in Saccharomyces cerevisiae in order to identify a transcription factor that mediates the effect of Tor1 on the expression of the ATGL ortholog in yeast. This factor, Msn4p, has homologues in mammalian cells that form a family of early growth response transcription factors. One member of the family, Egr1, is induced by insulin and nutrients and directly inhibits activity of the ATGL promoter in vitro and expression of ATGL in cultured adipocytes. Feeding animals a high-fat diet increases the activity of mTORC1 and the expression of Egr1 while decreasing ATGL levels in epididymal fat. We suggest that the evolutionarily conserved mTORC1-Egr1-ATGL regulatory pathway represents an important component of the antilipolytic effect of insulin in the mammalian organism.

Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression.

Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.

p130Cas acts as survival factor during PMA-induced apoptosis in HL-60 promyelocytic leukemia cells.

Phorbol 12-myristate 13-acetate (PMA) stimulates the differentiation of promyelocytic leukemia HL-60 cells by inducing adhesion followed by cell aggregation and, importantly, apoptosis. p130Cas (Crk-associated substrate) is an adapter molecule that controls cell growth, attachment and apoptotic programs. Notably, elevated p130Cas activity is associated with leukemias and lymphomas. Since p130Cas regulates cell adhesion, we tested the hypothesis that it participates in the differentiation of hematopoietic cells. Here we show that PMA mediates the late induction of p130Cas expression in HL-60 cells, which coincided with cell aggregation and the onset of apoptosis. Ectopic p130Cas expression led to increased cell adhesion and earlier cell aggregation potentially contributing to the observed increased cell viability in these transductants. p130Cas expression concurred with the induction of its own regulator the transcription factor EGR1, its coregulator NAB2, and apoptosis. NF-κB inhibition in PMA-treated HL-60 cells promoted the loss of cell aggregation and cell death. We further showed a reduction of p130Cas, EGR1, and NAB2 levels in response to NF-κB inhibition during PMA treatment. Hence, p130Cas acts as survival factor by limiting PMA-mediated cell cluster disruption and resulting cell death in HL-60 cells.

Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2.

Elevated levels of p130(Cas)/BCAR1 (Crk-associated substrate/breast cancer antiestrogen resistance 1) are found in aggressive breast tumors and are associated with tamoxifen resistance of mammary cancers. p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways frequently deregulated in breast cancer. To elucidate mechanisms leading to p130(Cas) up-regulation in mammary carcinomas and during acquired tamoxifen resistance, the regulation of p130(Cas)/BCAR1 was studied. Because multiple putative binding motifs for the inducible transcription factor EGR1 were identified in the 5' region of BCAR1, the p130(Cas)/BCAR1 regulation by EGR1 and its coregulator NAB2 was investigated. Overexpression or short interfering RNA (siRNA)-mediated down-regulation of EGR1 or NAB2, and chromatin immunoprecipitations indicated that EGR1 and NAB2 act in concert to positively regulate p130(Cas)/BCAR1 expression in breast cancer cells. p130(Cas) depletion using siRNA showed that, in tamoxifen-sensitive MCF-7 cells, p130(Cas) regulates EGR1 and NAB2 expression, whereas in the derivative tamoxifen-resistant TAM-R cells, only NAB2 levels were influenced. BCAR1 messenger RNA and p130(Cas) protein were upregulated by phorbol esters following the kinetics of late response genes in MCF-7 but not in TAM-R cells. Thus, in MCF-7 cells, we identified a positive feedback loop where p130(Cas) positively regulates EGR1 and NAB2, which in turn induce p130(Cas) expression. Importantly, compared with MCF-7, enhanced NAB2 expression and increased EGR1 binding to the BCAR1 5' region observed in TAM-R may lead to the constitutively increased p130(Cas)/BCAR1 levels in TAM-R cells. The uncovered differences in this EGR1/NAB2/p130(Cas) network in MCF-7 versus TAM-R cells may also contribute to p130(Cas) up-regulation during acquired tamoxifen resistance.

EGR1, EGR2, and EGR3 activate the expression of their coregulator NAB2 establishing a negative feedback loop in cells of neuroectodermal and epithelial origin.

The inducible zinc finger transcription factors EGR1, EGR2, and EGR3 regulate the expression of numerous genes involved in differentiation, growth, and response to extracellular signals. Their activity is modulated in part through NAB2 which is induced by the same stimuli. In melanoma and carcinoma cells EGR1 activates NAB2 expression. In T lymphocytes EGR2 and EGR3 have been shown to inhibit NAB2 expression. Therefore, we investigated the influence of EGR2 and EGR3 on NAB2 expression in melanoma and carcinoma cells. Here, we show that like EGR1, EGR2 and EGR3 induced NAB2 expression in these cells. EGR1 and EGR3 act in concert on the NAB2 promoter and are more potent activators of NAB2 transcription than EGR2. EGR1-, EGR2-, and EGR3-induced NAB2 promoter activity is mediated through similar cis-regulatory elements and the activation by each EGR is repressed by NAB2. Kinetic studies suggest that induction of EGR1 leads to low NAB2 expression, while EGR2 and EGR3 are necessary for maximal and sustained expression. As already shown for EGR1, reduction of EGR2 or EGR3 expression by siRNAs reduced endogenous NAB2 levels. Depletion of EGR3 also resulted in a reduction of EGR2 levels confirming EGR2 as a target gene of EGR3. Our results suggest that in many cells of neuroectodermal and epithelial origin EGR1, EGR2, and EGR3 activate NAB2 transcription which is in turn repressed by NAB2, thus establishing a negative feedback loop. This points to a complex relationship between the EGR factors and NAB2 expression likely depending on the cellular context.

Egr-1 induces the expression of its corepressor nab2 by activation of the nab2 promoter thereby establishing a negative feedback loop.

The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5'-region. The TATA- and initiator-less Nab2 promoter, located from -679 to -74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between -329 and -260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals.