An EMT-Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.National Institutes of Health (U.S.) (R01-HG002439)National Science Foundation (U.S.) (equipment grant)National Institutes of Health (U.S.) (Integrative Cancer Biology Program Grant U54-CA112967)David H. Koch Institute for Integrative Cancer Research at MIT (Ludwig Center for Metastasis Research)David H. Koch Institute for Integrative Cancer Research at MITMassachusetts Institute of Technology (Croucher Scholarship)Massachusetts Institute of Technology (Ludwig Fund postdoctoral fellowship)National Institutes of Health (U.S.) (NIH CA100324)National Institutes of Health (U.S.) (AECC9526-5267

Anti-glomerular basement membrane disease superimposed on membranous nephropathy: a case report and review of the literature

<p>Abstract</p> <p>Introduction</p> <p>Anti-glomerular basement membrane disease is a rare autoimmune disorder characterized by pulmonary hemorrhage, crescentic glomerulonephritis and the presence of circulating anti-glomerular basement membrane antibodies. The simultaneous occurrence of both anti-glomerular basement membrane disease and membranous nephropathy is rare.</p> <p>Case presentation</p> <p>A 59-year-old Hispanic man presented with acute onset of nausea and vomiting and was found to have renal insufficiency. Work-up included a kidney biopsy, which revealed anti-glomerular basement membrane disease with underlying membranous nephropathy. He was treated with emergent hemodialysis, intravenous corticosteroids, plasmapheresis, and cyclophosphamide without improvement in his renal function.</p> <p>Conclusion</p> <p>Simultaneous anti-glomerular basement membrane disease and membranous nephropathy is very rare. There have been 16 previous case reports in the English language literature that have been associated with a high mortality and morbidity, and a very high rate of renal failure resulting in hemodialysis. Co-existence of membranous nephropathy and anti-glomerular basement membrane disease may be immune-mediated, although the exact mechanism is not clear.</p

Membranous nephropathy and lupus-like syndrome after hematopoietic cell transplantation: a case report

<p>Abstract</p> <p>Introduction</p> <p>The kidney is increasingly recognised as a target organ of chronic graft-versus-host disease after hematopoietic cell transplantation in the context of the development of the nephrotic syndrome. Chronic graft-versus-host disease is associated with autoimmune phenomena similar, but not identical, to those observed in various rheumatologic disorders, implicating autoimmunity as an important component of the disease.</p> <p>Case presentation</p> <p>We report the case of a 57-year-old Caucasian man who developed the nephrotic syndrome due to membranous nephropathy in association with recurrent chronic graft-versus-host disease, along with a lupus-like syndrome manifested with pancytopenia, hair loss, positive anti-DNA antibodies and sub-epithelial and mesangial immune deposits. To the best of our knowledge, this is the first case reported in the literature. The nephrotic syndrome subsided soon after he was treated with a short course of cyclosporin with steroids. Unfortunately he died seven months later due to a relapse of leukemia.</p> <p>Conclusions</p> <p>Our case report confirms the notion that chronic graft-versus-host disease is characterized by the appearance of autoimmune phenomena similar, but not identical, to those seen in autoimmune diseases. The decision for more immunosuppression has to be weighed against the need for preservation of the graft versus leukemia phenomenon.</p

Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

<p>Abstract</p> <p>Background</p> <p><it>HER2 </it>gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence <it>in situ </it>hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods.</p> <p>Methods</p> <p>In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms.</p> <p>Results</p> <p>Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to "ratio skewing" caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability.</p> <p>Conclusions</p> <p>These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two probe FISH interphase analysis is inadequate and results interpreted using the HER2/CEP17 ratio should be reported "with caution" when the presence of centromeric amplification or monosomy is suspected by FISH signal gains or losses. The presence of these pericentromeric copy number changes may result in artificial skewing of the HER2/CEP17 ratio towards false negative or false positive results in breast cancer with chromosome 17 complexity. Full genomic analysis should be considered in all cases with complex chromosome 17 aneusomy as these cases are likely to have genome-wide instability, amplifications, and a poor prognosis.</p

Abstract P2-08-03: Phosphatidylinositol-3-kinase mutations are common in lobular neoplasia

Abstract The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated in invasive breast carcinoma, with PIK3CA mutations present in ∼25% of invasive carcinomas, and several studies demonstrating an even higher prevalence of PIK3CA mutations in invasive lobular carcinomas. Lobular neoplasia (LN), including lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH), are controversial lesions that may represent non-obligate precursor of invasive lobular carcinoma. However, lobular neoplasia has not yet been systematically studied for activating point mutations. Twenty-six breast resection specimens containing LN and/or invasive lobular carcinoma (ILC) were identified from the files of Oregon Health &amp; Science University. Adjacent lesions including columnar cell change (CCC), usual ductal hyperplasia (UDH), invasive or in-situ ductal carcinoma, and lymph node metastases were separately isolated where available. DNA was prepared from punches of formalin-fixed paraffin-embedded tissue blocks using standard methods. DNA extracts were screened for a panel of point mutations using a multiplex PCR panel with a mass-spectroscopy readout (Sequenom MassArray). The panel covers 643 point mutations in 53 genes including AKT1/2/3, ALK, BRAF, CDK4, CSF1R, CTNNB1, EGFR, ERBB2, ERCC6, FBX4, FBXW7, FES, FGFR1/2/3/4, FOXL2, GNA11, GNAQ, GNAS, HRAS, IDH1/2, IGF1R, KDR, KIT, KRAS, MAPK2K1/2/7, MET, MYC, NEK9, NRAS, NTRK1/2/3, PDGFRA, PIK3CA, PIK3R1/4/5, PKHD1, PRKCB1, RAF1, RET, SMO, SOS1, STAT1, TEC, and TP53; covering 41 substitutions in 23 codons of the PIK3CA gene. PIK3CA mutations were identified in 8/22 LN (36%; PIK3CA exon 4 N345K-1; exon 9 E542K-1; E545K-3; exon 20 H1047R-3), and in 11/16 ILC (68%; PIK3CA exon 4 N345K-2; exon 9 E542K-1, E545K-2, Q546R-1; exon 20 H1047L-1, H1047R-4, one with concomitant HRAS G12D mutation). LN and coincident ILC were tested in 11 patients; 4 patients had the same point mutations in LN and ILC (concordant mutant); 4 patients were wildtype for all codons tested in LN and ILC (concordant wildtype); 3 patients had discordant mutation status (LCIS-E545K/ILC-H1047R; LCIS-E542K/ILC-H1047R; ALH-WT/ILC-H1047R &amp; HRAS G12V). Four patients had LN and invasive ductal carcinoma (IDC); of these, 2 were discordant (LCIS-H1047R/IDC-wildtype; ALH-wildtype/IDC-E545K) and 2 concordant wildtype. Two additional patients had discrete IDC and ILC tumors; in both the IDC was wildtype, but the ILC harbored a PIK3CA mutation. Concurrent CCC and UDH were also screened, yielding 10/22 (45%) lesions with PIK3CA point mutations; in 3 instances the UDH mutation was concordant with LN/ILC, whereas CCC from the same specimen has discordant mutational status. Our study confirms the high prevalence of PIK3CA hotspot point mutations in ILC (68%). Importantly, we screened LCIS and ALH for a large panel of point mutations, and found only PIK3CA mutations (36% of lesions). Although a small cohort, the mutation status of concurrent LN and ILC was frequently concordant (8/11=72%), and in fact, quite similar to the degree of mutational concordance between paired DCIS and IDC in the literature and in our own experience (66–77%). This provides some support to the notion of LN as a precursor to ILC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-08-03.</jats:p

Regulatory function of trefoil peptides (TFF) on intestinal cell junctional complexes

Trefoil peptides (TFF) are constitutively expressed in the gastrointestinal tract and are involved in gastrointestinal defence and repair by promoting epithelial restitution. Although there is a general consensus regarding the pro-motogenic activity of trefoil peptides, the cellular mechanisms through which they mediate these processes are not completely understood. Pertubation of the E-cadherin/catenin complex at intercellular junctions appears to be a functional pathway through which TFF2 and TFF3 promote cell migration. Tight junction complexes seal the paracellular spaces between cells and contribute to epithelial barrier function. TFF3 peptide stimulation stabilises these junctions through upregulation of the tightening protein claudin-1 and redistribution of ZO-1 from the cytoplasm to the intercellular membrane with an increase in binding to occludin. Modulation of the functional activity and subcellular localisation of epithelial junctional adhesion molecules represent important mechanisms by which trefoil peptides may promote migration of intestinal epithelial cells in vitro and healing of mucosal damage in vivo

Recombinant human G-CSF increases invasiveness of breast cancer cells.

Abstract Abstract #4057 Background: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) is commonly given to breast cancer patients to prevent neutropenia during chemotherapy. It binds to the G-CSF receptor (G-CSFR) on neutrophil precursors, promoting their proliferation within the bone marrow. We observed breast cancer growth in patients with advanced breast cancer during rhG-CSF administration. The G-CSFR is present on some solid tumors, but has not been examined in breast cancer. We asked whether breast cancer cells express the G-CSFR, and whether rhG-CSF has any direct effects on breast cancer proliferation or invasion in vitro.&amp;#x2028; Material and Methods: MDA MB231 is a highly aggressive ER–PR–Her2/neu– breast cancer cell line derived from a human pleural effusion. We analyzed this cell line for the presence of the G-CSFR by immunohistochemistry, reverse-transcriptase PCR (RT-PCR), Western Blot analysis and flow cytometry. We sequenced DNA products of RT-PCR to verify results. We examined the effects of physiologic doses of rhG-CSF on breast cancer cell proliferation and invasion using MTT proliferation and Matrigel transwell assays, respectively.&amp;#x2028; Results: MDA MB231 breast cancer cells express G-CSFR protein and mRNA. Of seven known G-CSFR isoforms, we found evidence of isoforms 1 and 4. Treating MDA MB231 breast cancer cells with rhG-CSF at 50ng/ml did not increase cell proliferation, but did increase invasion across a Matrigel-coated transwell membrane. Cells treated with rhG-CSF appeared more stellate than untreated cells. Studies are ongoing to evaluate which signal transduction pathways and genes are involved in this increased invasion response. The effects of rhG-CSF on breast cancer growth in vivo are also being examined in murine models.&amp;#x2028; Discussion: rhG-CSF is given routinely as part of many adjuvant chemotherapy regimens for breast cancer. Although dose-dense regimens using rhG-CSF have shown equivalent or improved survival when compared to standard dosing regimens, it is possible that rhG-CSF may increase the growth or invasive potential of some breast cancers. It is also possible that rhG-CSF could mobilize breast cancer cells from the bone marrow, making these cells more susceptible to chemotherapy. These possibilities should promote thoughful use of rhG-CSF in the clinical setting, and should factor into the design and implementation of clinical trials using rhG-CSF. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4057.</jats:p