Die Rolle des EGR/NAB2/p130Cas Signaltransduktions-Netzwerks in der Progression und Therapieresistenzentwicklung von Tumoren – Identifikation von Biomarkern und therapeutischen Zielen

Das breast cancer antiestrogen resistance 1 (BCAR1) -Gen kodiert für das Adapterprotein p130 Crk-associated substrate (Cas), welches als Knotenpunkt für Signalprotein-Komplexe dient, die Signaltransduktionswege kontrollieren, welche in Tumorzellen häufig dereguliert sind. Unter anderem interagiert p130Cas mit den Kinasen der Src-Familie, der focal adhesion kinase (FAK) und phosphoinositide 3-kinase (PI3K) und ist dadurch an der Kontrolle der Aktivität der rat sarcoma (RAS) /extracellular signal regulated kinase (ERK), PI3K und c-Jun N-terminal kinases (JNK) Signalwege beteiligt. Eine starke Überexpression oder Überaktivierung von p130Cas, die mit Tumorprogression und Therapieresistenz einherging, wurde zunächst in Mammakarzinomen und in den letzten Jahren auch in anderen Tumorentitäten beschrieben. Im Rahmen des Habilitationsprojektes wurden die Mechanismen der Überexpression von p130Cas und der damit assoziierten Therapie-Resistenz-Entstehung in verschiedenen Krebsentitäten untersucht. Außerdem wurden Ansätze zur zielgerichteten p130Cas-Inhibition auf ihre Wirksamkeit geprüft. Es konnte ein Regulations-Netzwerk aus den Transkriptionsfaktoren der early growth response (EGR) -Familie, des EGR-Ko-Regulators NGFI-A binding protein 2 (NAB2) und p130Cas identifiziert werden. Vor allem die starke NAB2-Expression konnte als eine der Ursachen der konstitutiven p130Cas-Expression in Anti-Östrogen-resistenten Zellen/Patientenproben ausgemacht werden. Des Weiteren konnte die Bedeutung des EGR/NAB2/p130Cas-Systems bei der Antwort auf Differenzierungstherapie in Kombination mit NF-B-Inhibition in einem Modell der Akuten Promyelozyten Leukämie (APL) gezeigt werden. Hier scheint p130Cas als Überlebensfaktor zu wirken. In kolorektalen Karzinomen (KRK) wurde ein signifikant erhöhtes Sterberisiko bei BCAR1-exprimierenden rechtsseitigen, Stadium I/II, MSS oder BRAF-mutierten KRK gefunden. Außerdem konnte durch Herunterregulierung der BCAR1/p130Cas-Expression ein verbessertes Ansprechen auf FOLFIRI (Folinsäure/5-FU/Irinotecan) erreicht werden. Diese Ergebnisse verdeutlichen die Rolle von p130Cas und NAB2 als therapeutische Ziele bei Hormonrezeptor-positivien (HR+) Mammakarzinomen und APL, sowie von p130Cas als Prognosefaktor und Behandlungsziel bei bestimmten Subtypen von KRK. Ein weiterer Aspekt der Habilitationsarbeit war die Identifikation von neuen Biomarkern und therapeutischen Zielen für Fernmetastasen bei prämenopausalen Patientinnen mit frühem Hormon-Rezeptor-positivem (HR+) /HER2-negativem Brustkrebs. Durch RNA-Expressionsprofile und Korrelationen mit klinischen Parametern konnten vier Signaturen (risk of recurrence (ROR), progesteron receptor, claudin-low und mammary stemness) mit dem Metastasen-freien Überleben assoziiert und LDL receptor related protein 2 (LRP2), integrin binding Sialoprotein (IBSP) und signal peptide, CUB domain, and EGF-like domain-containing 2 (SCUBE2) als unabhängige prognostische Faktoren identifiziert werden. Zusammenfassend konnte die Rolle von p130Cas als therapeutisches Ziel, als Resistenz-vermittelnder Faktor und als prognostischer Biomarker in verschiedenen Malignitäten bekräftigt werden. Des Weiteren konnten prognostische Gensignaturen und Einzelgenmarker für Fernmetastasen bei prämenopausalem frühen HR+/HER2- Brustkrebs identifiziert werden, die in der zukünftigen klinischen Praxis Anwendung finden könnten

Complement membrane attack and tumorigenesis: a systems biology approach

Tumor development driven by inflammation is now an established phenomenon, but the role that complement plays remains uncertain. Recent evidence has suggested that various components of the complement (C) cascade may influence tumor development in disparate ways; however, little attention has been paid to that of the membrane attack complex (MAC). This is despite abundant evidence documenting the effects of this complex on cell behavior, including cell activation, protection from/induction of apoptosis, release of inflammatory cytokines, growth factors, and ECM components and regulators, and the triggering of the NLRP3 inflammasome. Here we present a novel approach to this issue by using global gene expression studies in conjunction with a systems biology analysis. Using network analysis of MAC-responsive expression changes, we demonstrate a cluster of co-regulated genes known to have impact in the extracellular space and on the supporting stroma and with well characterized tumor-promoting roles. Network analysis highlighted the central role for EGF receptor activation in mediating the observed responses to MAC exposure. Overall, the study sheds light on the mechanisms by which sublytic MAC causes tumor cell responses and exposes a gene expression signature that implicates MAC as a driver of tumor progression. These findings have implications for understanding of the roles of complement and the MAC in tumor development and progression, which in turn will inform future therapeutic strategies in cancer

The DNA-polymorphism rs849142 is associated with skin toxicity induced by targeted anti-EGFR therapy using cetuximab

Skin toxicity (ST) is a frequent adverse effect (AE) in anti-epidermal growth factor receptor (EGFR)-targeted treatment of metastatic colorectal cancer (mCRC) resulting in decreased quality of life and problems in clinical management. We wanted to identify biomarkers predicting ST in this setting and focused on 70 DNA polymorphisms associated with acne, the (immunoglobulin fragment crystallizable region) Fcγ-receptor pathway, and systemic lupus erythematosus (SLE) applying next-generation-sequencing (NGS). For the analysis patients with mCRC treated with cetuximab were selected from the FIRE-3 study. A training group consisting of the phenotypes low (1) - and high-grade (3) ST (n = 16) and a validation group (n = 55) representing also the intermediate grade (2) were genotyped and investigated in a genotype-phenotype association analysis. The single nucleotide polymorphism (SNP) rs849142 significantly associated with ST in both the training- (p < 0.01) and validation-group (p = 0.04). rs849142 is located in an intron of the juxtaposed with another zinc finger protein 1 (JAZF1) gene. Haplotype analysis demonstrated significant linkage disequilibrium of rs849142 with JAZF1. Thus, rs849142 might be a predictive biomarker for ST in anti-EGFR treated mCRC patients. Its value in the clinical management of AE has to be validated in larger cohorts

Mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon: detailed molecular characterisation of two cases indicates a distinct colorectal cancer entity

We present two rare cases of mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon. A literature search revealed only three published cases with similar histology but none of these reports provided profound molecular and mutational analyses. Our two cases exhibited a distinct, colon-like immunophenotype with strong nuclear CDX2 and beta-catenin expression in more than 90% of the tumour cells of both components. We analysed the two carcinomas regarding microsatellite stability, RAS, BRAF and PD-L1 status. In addition, next-generation panel sequencing with Ion AmpliSe (TM) psi Cancer Hotspot Panel v2 was performed. This approach revealed mutations in FBXW7, CTNNB1 and PIK3CA in the first case and FBXW7 and RB1 mutations in the second case. We looked for similar mutational patterns in three publicly available colorectal adenocarcinoma data sets, as well as in collections of colorectal mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) and colorectal neuroendocrine carcinomas. This approach indicated that the FBXW7 point mutation, without being accompanied by classical adenoma-carcinoma sequence mutations, such as APC, KRAS and TP53, likely occurs at a relatively high frequency in mixed neuroendocrine and squamous cell carcinoma and therefore may be characteristic for this rare tumour type. FBXW7 codifies the substrate recognition element of an ubiquitin ligase, and inactivating FBXW7 mutations lead to an exceptional accumulation of its target beta-catenin which results in overactivation of the Wnt-signalling pathway. In line with previously described hypotheses of de-differentiation of colon cells by enhanced Wnt-signalling, our data indicate a crucial role for mutant FBXW7 in the unusual morphological switch that determines these rare neoplasms. Therefore, mixed large cell neuroendocrine and a squamous cell carcinoma can be considered as a distinct carcinoma entity in the colon, defined by morphology, immunophenotype and distinct molecular genetic alteration(s)

Biomarker alterations associated with distinct patterns of metastatic spread in colorectal cancer

Metastatic spread is the most important life-threatening feature of colorectal cancer and is supposed to be mainly driven by alterations in different carcinogenic pathways. The present study compared mutation and expression profiles of distinctive biomarkers in colorectal cancer patients with different clinical metastatic patterns. As for a case-control study, patients were matched according to T category, grading and primary tumour site. Overall, 246 patients with either exclusive lung metastasis (N = 82), exclusive liver metastasis (N = 82) or non-metastatic colorectal cancer (N = 82) were identified. Paraffin-embedded specimens were examined for mutations in the RAS and RAF genes and for the expression of β-catenin and CD133. Clinical endpoints were presence or absence of distant metastasis, formation of metastasis in lungs versus the liver and survival. MAPK pathway mutations in either the KRAS, NRAS or BRAF gene were associated with the development of lung metastasis (63.4%) compared to the control group (47.6%; p = 0.04). MAPK pathway alterations plus high β-catenin expression were associated with metastasis to the lungs but not to the liver (28.0% vs. 13.4%; p = 0.02). High CD133 expression correlated with the development of liver metastasis compared to the control group (30.5% vs. 14.6%; p = 0.02). This data indicates that different patterns of distant spread are associated with specific biomarker alterations and may represent different molecular subtypes of colorectal cancer. However, underlying mechanisms of metastasis formation in different anatomic sites remains unclear. Since knowledge of the anticipated site of distant spread would substantially impact clinical management, further research is needed to identify solid biomarkers for different metastatic patterns

Proteomics uncover EPHA2 as a potential novel therapeutic target in colorectal cancer cell lines with acquired cetuximab resistance.

BACKGROUND: In metastatic colorectal cancer (mCRC), acquired resistance against anti-EGFR targeted monoclonal antibodies, such as cetuximab (CET), was shown to be frequently caused by activating alterations in the RAS genes KRAS or NRAS. To this day, no efficient follow-up treatment option has emerged to treat mCRC in such a setting of resistance. METHODS: To uncover potential targets for second-line targeted therapies, we used mass-spectrometric proteomics to shed light on kinome reprogramming in an established cellular model of acquired, KRAS-associated CET resistance. RESULTS: This CET resistance was reflected by significant changes in the kinome, most of them individual to each cell line. Interestingly, all investigated resistant cell lines displayed upregulation of the Ephrin type-A receptor 2 (EPHA2), a well-known driver of traits of progression. Expectedly resistant cell lines displayed increased migration (p < 0.01) that was significantly reduced by targeting the EPHA2 signalling axis using RNA interference (RNAi) (p < 0.001), ephrin-A1 stimulation (p < 0.001), dasatinib (p < 0.01), or anti-EPHA2 antibody treatment (p < 0.001), identifying it as an actionable target in mCRC with acquired CET resistance. CONCLUSION: These results highlight EPHA2 and its role in mCRC with KRAS-gene mutated acquired CET resistance and support its use as a potential actionable target for the development of future precision medicine therapies

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)