Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines

<p>Abstract</p> <p>Background</p> <p>Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice.</p> <p>Results</p> <p>Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in <it>Gli1</it>^-/-<it>2</it>^-/-iMEFs was severely reduced while <it>Gli2</it>^-/-<it>3</it>^-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both <it>Gli1</it>^-/-<it>2</it>^-/-and <it>Gli2</it>^-/-<it>3</it>^-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.</p> <p>Conclusion</p> <p>This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.</p

Editorial: translational insights into pancreatic ductal adenocarcinoma

No abstract available

Impact of the COVID-19 pandemic on breast cancer incidence and tumor stage in the Netherlands and Norway:A population-based study

BACKGROUND: Comparing the impact of the COVID-19 pandemic on the incidence of newly diagnosed breast tumors and their tumor stage between the Netherlands and Norway will help us understand the effect of differences in governmental and social reactions towards the pandemic.METHODS: Women newly diagnosed with breast cancer in 2017-2021 were selected from the Netherlands Cancer Registry and the Cancer Registry of Norway. The crude breast cancer incidence rate (tumors per 100,000 women) during the first (March-September 2020), second (October 2020-April 2021), and Delta COVID-19 wave (May-December 2021) was compared with the incidence rate in the corresponding periods in 2017, 2018, and 2019. Incidence rates were stratified by age group, method of detection, and clinical tumor stage.RESULTS: During the first wave breast cancer incidence declined to a larger extent in the Netherlands than in Norway (27.7% vs. 17.2% decrease, respectively). In both countries, incidence decreased in women eligible for screening. In the Netherlands, incidence also decreased in women not eligible for screening. During the second wave an increase in the incidence of stage IV tumors in women aged 50-69 years was seen in the Netherlands. During the Delta wave an increase in overall incidence and incidence of stage I tumors was seen in Norway.CONCLUSION: Alterations in breast cancer incidence and tumor stage seem related to a combined effect of the suspension of the screening program, health care avoidance due to the severity of the pandemic, and other unknown factors.</p

Ccaat/enhancer-binding protein delta (C/ebpδ): A previously unrecognized tumor suppressor that limits the oncogenic potential of pancreatic ductal adenocarcinoma cells

CCAAT/enhancer-binding protein δ (C/EBPδ) is a transcription factor involved in growth arrest and differentiation, which has consequently been suggested to harbor tumor suppressive activities. However, C/EBPδ over-expression correlates with poor prognosis in glioblastoma and promotes genomic instability in cervical cancer, hinting at an oncogenic role of C/EBPδ in these contexts. Here, we explore the role of C/EBPδ in pancreatic cancer. We determined C/EBPδ expression in biopsies from pancreatic cancer patients using public gene-expression datasets and in-house tissue microarrays. We found that C/EBPδ is highly expressed in healthy pancreatic ductal cells but lost in pancreatic ductal adenocarcinoma. Furthermore, loss of C/EBPδ correlated with increased lymph node involvement and shorter overall survival in pancreatic ductal adenocarcinoma patients. In accordance with this, in vitro experiments showed reduced clonogenic capacity and proliferation of pancreatic ductal adenocarcinoma cells following C/EBPδ re-expression, concurrent with decreased sphere formation capacity in soft agar assays. We thus report a previously unrecognized but important tumor suppressor role of C/EBPδ in pancreatic ductal adenocarcinoma. This is of particular interest since only few tumor suppressors have been identified in the context of pancreatic cancer. Moreover, our findings suggest that restoration of C/EBPδ activity could hold therapeutic value in pancreatic ductal adenocarcinoma, although the latter claim needs to be substantiated in future studies

Repression of Smoothened by Patched-Dependent (Pro-)Vitamin D3 Secretion

The developmentally important hedgehog (Hh) pathway is activated by binding of Hh to patched (Ptch1), releasing smoothened (Smo) and the downstream transcription factor glioma associated (Gli) from inhibition. The mechanism behind Ptch1-dependent Smo inhibition remains unresolved. We now show that by mixing Ptch1-transfected and Ptch1 small interfering RNA–transfected cells with Gli reporter cells, Ptch1 is capable of non–cell autonomous repression of Smo. The magnitude of this non–cell autonomous repression of Smo activity was comparable to the fusion of Ptch1-transfected cell lines and Gli reporter cell lines, suggesting that it is the predominant mode of action. CHOD-PAP analysis of medium conditioned by Ptch1-transfected cells showed an elevated 3β-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3β-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3β-hydroxysteroid (pro-)vitamin D3 to the medium effectively inhibited Gli activity. Vitamin D3 bound to Smo with high affinity in a cyclopamine-sensitive manner. Treating zebrafish embryos with vitamin D3 mimicked the smo (–/–) phenotype, confirming the inhibitory action in vivo. Hh activates its signalling cascade by inhibiting Ptch1-dependent secretion of the 3β-hydroxysteroid (pro-)vitamin D3. This action not only explains the seemingly contradictory cause of Smith-Lemli-Opitz syndrome (SLOS), but also establishes Hh as a unique morphogen, because binding of Hh on one cell is capable of activating Hh-dependent signalling cascades on other cells

Serum-based measurements of stromal activation through ADAM12 associate with poor prognosis in colorectal cancer.

BACKGROUND Recently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal activation, A Disintegrin and Metalloprotease 12 (ADAM12) in colorectal cancer (CRC). METHODS Using gene expression databases we investigated ADAM12 expression in CRC and delineated the source of ADAM12 expression. The clinical value of ADAM12 was retrospectively assessed in the CAIRO2 trial in metastatic CRC with 235 patients (31% of total cohort), and an independent rectal cancer cohort (n = 20). RESULTS ADAM12 is expressed by activated CRC associated fibroblasts. In the CAIRO2 trial cohort, ADAM12 serum levels were prognostic (ADAM12 low versus ADAM12 high; median OS 25.3 vs. 17.1 months, HR 1.48 [95% CI 1.11-1.96], P = 0.007). The prognostic potential was specifically high for metastatic rectal cancer (HR 1.78 [95% CI 1.06-3.00], P = 0.030) and mesenchymal subtype tumors (HR 2.12 [95% CI 1.25-3.60], P = 0.004). ADAM12 also showed potential for predicting recurrence in an exploratory analysis of non-metastatic rectal cancers. CONCLUSIONS Here we describe a non-invasive marker for activated stroma in CRC which associates with poor outcome, especially for primary cancers located in the rectum

Spatiotemporal regulation of clonogenicity in colorectal cancer xenografts

Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system

Phase II Feasibility and Biomarker Study of Neoadjuvant Trastuzumab and Pertuzumab With Chemoradiotherapy for Resectable Human Epidermal Growth Factor Receptor 2-Positive Esophageal Adenocarcinoma:TRAP Study

PURPOSE: Approximately 15% to 43% of esophageal adenocarcinomas (EACs) are human epidermal growth factor receptor 2 (HER2) positive. Because dual-agent HER2 blockade demonstrated a survival benefit in breast cancer, we conducted a phase II feasibility study of trastuzumab and pertuzumab added to neoadjuvant chemoradiotherapy (nCRT) in patients with EAC. PATIENTS AND METHODS: Patients with resectable HER2-positive EAC received standard nCRT with carboplatin and paclitaxel and 41.4 Gy of radiotherapy, with 4 mg/kg of trastuzumab on day 1, 2 mg/kg per week during weeks 2 to 6, and 6 mg/kg per week during weeks 7, 10, and 13 and 840 mg of pertuzumab every 3 weeks. The primary end point was feasibility, defined as ≥ 80% completion of treatment with both trastuzumab and pertuzumab. An exploratory comparison of survival with a propensity score-matched cohort receiving standard nCRT was performed, as were exploratory pharmacokinetic and biomarker analyses. RESULTS: Of the 40 enrolled patients (78% men; median age, 63 years), 33 (83%) completed treatment with trastuzumab and pertuzumab. No unexpected safety events were observed. R0 resection was achieved in all patients undergoing surgery, with pathologic complete response in 13 patients (34%). Three-year progression-free and overall survival (OS) were 57% and 71%, respectively (median follow-up, 32.1 months). Compared with the propensity score-matched cohort, a significantly longer OS was observed with HER2 blockade (hazard ratio, 0.58; 95% CI, 0.34 to 0.97). Results of pharmacokinetic analysis and activity on [18F]fluorodeoxyglucose positron emission tomography scans did not correlate with survival or pathologic response. Patients with HER2 3+ overexpression or growth factor receptor-bound protein 7 (Grb7) -positive tumors at baseline demonstrated significantly better survival (P = .007) or treatment response (P = .016), respectively. CONCLUSION: Addition of trastuzumab and pertuzumab to nCRT in patients with HER2-positive EAC is feasible and demonstrates potentially promising activity compared with historical controls. HER2 3+ overexpression and Grb7 positivity are potentially predictive for survival and treatment response, respectively