NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications

INTRODUCTION: Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells. METHODS: Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both. RESULTS: Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G(1 )phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo. CONCLUSIONS: Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes

Notch versus the proteasome: what is the target of γ-secretase inhibitor-I?

γ-Secretase inhibitors are new anti-cancer agents targeting Notch signaling. Their specificity for Notch is as yet unclear. Han and colleagues investigated the effects of Z-LeuLeuNleu-CHO on growth of breast cancer cells. The results demonstrated a reduction in cell viability primarily via proteasome inhibition independent of Notch activity. Currently, γ-secretase inhibitors in clinical trials are structurally distinct from Z-LeuLeuNleu-CHO. Their effects on the proteasome are yet to be determined. However, findings from Han and colleagues pose two critical questions: Is the level of proteasomal activity in breast tumors the driving force for growth? What does the Notch pathway contribute to this growth

New Perspectives on Diagnosis and Therapy of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare, but severe form of cancer, with an incidence that varies significantly within and among different countries around the world. It develops in about one to two persons per million of the general population, leading to thousands of deaths every year worldwide. To date, the MPM is mostly associated with occupational asbestos exposure. Asbestos represents the predominant etiological factor, with approximately 70% of cases of MPM with well-documented occupational exposure to asbestos, with the exposure time, on average greater than 40 years. Environmental exposure to asbestos is increasingly becoming recognized as a cause of mesothelioma, together with gene mutations. The possible roles of other cofactors, such as viral infection and radiation exposure, are still debated. MPM is a fatal tumor. This cancer arises during its early phase without clinical signs. Consequently, its diagnosis occurs at advanced stages. Standard clinical therapeutic approaches include surgery, chemo- and radiotherapies. Preclinical and clinical researches are making great strides in the field of this deadly disease, identifying new biomarkers and innovative therapeutic approaches. Among the newly identified markers and potential therapeutic targets, circulating microRNAs and the Notch pathway represent promising avenues that could result in the early detection of the tumor and novel therapeutic approaches

Collagen XV Inhibits Epithelial to Mesenchymal Transition in Pancreatic Adenocarcinoma Cells

<div><p>Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth <i>in vitro</i> and to reduce tumor burden and increase survival <i>in vivo</i> support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated <i>in vitro</i> by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.</p></div

Collagen XV inhibits phosphorylation of Pyk2 and suppresses N-Cadherin.

<p>Vector-only (BxVC3, BxVC4, BxVC5, BxVC10) and COLXV-expressing clones (Bx15.5, Bx15.14, Bx15.23, Bx15.24) were grown on COLI coated plates (A, B,); plastic and COLI (C) or plastic alone (F) and lysed after 48 h. Western blots probed with antibodies specific for A) total FAK or pFAK; B) total Pyk2 or pPyk2; C, F) N-Cad; are shown. ß-tubulin provides an estimate of total protein. Expression of COLXV slightly enhances pFAK relative to FAK (though this is not statistically significant, p = 0.0913), but depresses pPyk2 relative to Pyk2 (* p = 0.0447) and suppresses N-Cadherin levels. Blots were quantified by integrating data acquired with the Lasso tool of Adobe Photoshop (C, D, E, F) and analyzed by students t test (E, *p<0.01). Each clone was analyzed at least 3 times for FAK, pFAK, Pyk2, pPyk2 and N-Cad expression.</p

Collagen XV inhibits scatter of BxPC-3 cells on a collagen I substrate and interacts directly with DDR1 and E-Cadherin.

<p>A) Vector control (BxVC3) and COLXV expressing (Bx15.1, Bx15.14) clones are shown grown on plastic substrate and on COLI coated substrate. Phase contrast microscopy, all panels 100X magnification. 3 vector clones and 5 COLXV expressing clones (2 with low expression and 3 with high expression) were analyzed at least 3 times and the results were consistent. B) Immunoprecipitation (IP) of DDR1 from vector clone (BxVC3) and COLXV clone (Bx15.14), followed by probing of western blots of the IP material with antibodies specific for COLXV, DDR1 and E-Cad. C) IP of FLAG-tagged COLXV (FCOLXV) from the BxF15.3 clone using the M2 antibody. D) IP of COLXV from clone Bx15.3 with M2 after depletion of DDR1 with a specific siRNA (DDR1i) or transfection of scrambled control siRNA (Scrbi). Depletion of DDR1 reduced the amount of DDR1, but not E-Cad, interacting with COLXV. E) IP of COLXV from clone BxF15.3 with M2 after depletion of E-Cad with a specific siRNA (E-Cadi) or transfection of scrambled control siRNA (Scrbi). Depletion of E-Cad reduced the amounts interacting with COLXV. All experiments were performed a minimum of 3 times with consistent results.</p

E-Cadherin is stabilized at the cell periphery in collagen XV expressing cells.

<p>Confocal microscopy with an antibody specific for the extracellular domain of E-Cad (green) and nuclei stained with DAPI (blue). BxVC1 vector clone and Bx15.23 COLXV clone grown on plastic A) or COLI B). E-Cad is most abundant at the cell surface in both clones on plastic. On COLI, E-Cad moves from the cell periphery into the cytoplasm in BxVC1, but this redistribution is inhibited in the presence of COLXV (BX15.23). C) EEA1 (red) is found in the endoplasmic reticulum (ER)/Golgi zone of the cells grown on plastic, while E-Cad is at the cell periphery. D) After relocation of E-Cad on COLI, EEA1 colocalizes with E-Cad (white arrowheads) in BxVC1 cells but not Bx15.23 cells. Images are representative of several clones. E) Flow cytometry after staining cells with an E-Cad antibody shows increased cell-surface expression of E-Cad in cells with COLXV (Bx15.5 and 15.24) in comparison to vector controls (BxVC4 and BxVC1). All experiments performed a minimum of 3 times with consistent results.</p