Galina Khramtsova

Wnt/β-Catenin Pathway Activation Is Enriched in Basal-Like Breast Cancers and Predicts Poor Outcome

Although Wnt/beta-catenin pathway activation has been implicated in mouse models of breast cancer, there is contradictory evidence regarding its importance in human breast cancer. In this study, invasive and in situ breast cancer tissue microarrays containing luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)(+)/ER(-) and basal-like breast cancers were analyzed for beta-catenin subcellular localization. We demonstrate that nuclear and cytosolic accumulation of beta-catenin, a read-out of Wnt pathway activation, was enriched in basal-like breast cancers. In contrast, membrane-associated beta-catenin was observed in all breast cancer subtypes, and its expression decreased with tumor progression. Moreover, nuclear and cytosolic localization of beta-catenin was associated with other markers of the basal-like phenotype, including nuclear hormone receptor and HER2 negativity, cytokeratin 5/6 and vimentin expression, and stem cell enrichment. Importantly, this subcellular localization of beta-catenin was associated with a poor outcome and is more frequently observed in tumors from black patients. In addition, beta-catenin accumulation was more often observed in basal-like in situ carcinomas than other in situ subtypes, suggesting that activation of this pathway might be an early event in basal-like tumor development. Collectively, these data indicate that Wnt/beta-catenin activation is an important feature of basal-like breast cancers and is predictive of worse overall survival, suggesting that it may be an attractive pharmacological target for this aggressive breast cancer subtype.

TNFR2 activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated barrier loss and experimental colitis.

BACKGROUND & AIMS Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease. However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are not completely understood. We investigated these processes using colitis models in mice. METHODS We induced colitis by adoptive transfer of CD4(+)CD45RB(hi) cells or administration of dextran sulfate sodium to mice, including those deficient in tumor necrosis factor receptor (TNFR) 1, TNFR2, or the long isoform of myosin light chain kinase (MLCK). Intestinal tissues and isolated epithelial cells were analyzed by immunoblot, immunofluorescence, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction assays. RESULTS Induction of immune-mediated colitis by CD4(+)CD45RB(hi) adoptive transfer increased intestinal permeability, epithelial expression of claudin-2, the long isoform of MLCK, and TNFR2 (but not TNFR1) and phosphorylation of the myosin II light chain. Long MLCK upregulation, myosin II light chain phosphorylation, barrier loss, and weight loss were attenuated in TNFR2(-/-) , but not TNFR1(-/-) , recipients of wild-type CD4(+)CD45RB(hi) cells. Similarly, long MLCK(-/-) mice had limited increases in myosin II light chain phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of adoptive transfer-induced colitis. However, coincident with onset of epithelial apoptosis, long MLCK(-/-) mice ultimately developed colitis. This indicates that disease progresses via apoptosis in the absence of MLCK-dependent tight junction regulation. In support of this conclusion, long MLCK(-/-) mice were not protected from epithelial apoptosis-mediated, damage-dependent dextran sulfate sodium colitis. CONCLUSIONS In immune-mediated inflammatory bowel disease models, TNFR2 signaling increases long MLCK expression, resulting in tight junction dysregulation, barrier loss, and induction of colitis. At advanced stages, colitis progresses by apoptosis and mucosal damage that result in tight junction- and MLCK-independent barrier loss. Therefore, barrier loss in immune-mediated colitis occurs via two temporally and morphologically distinct mechanisms. Differential targeting of these mechanisms can lead to improved inflammatory bowel disease therapies.

Regulation of Tcf7l1 DNA Binding and Protein Stability as Principal Mechanisms of Wnt/β-Catenin Signaling

Wnt/β-catenin signal transduction requires direct binding of β-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. β-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-β-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/β-catenin in breast cancer.

Genome-wide profiles of CtBP link metabolism with genome stability and epithelial reprogramming in breast cancer

The C-terminal binding protein (CtBP) is a NADH-dependent transcriptional repressor that links carbohydrate metabolism to epigenetic regulation by recruiting diverse histone modifying complexes to chromatin. Here, global profiling of CtBP in breast cancer cells reveals that it drives epithelial to mesenchymal transition, stem cell pathways, and genome instability. CtBP expression induces mesenchymal and stem cell-like features while CtBP depletion or caloric restriction reverses gene repression and increases DNA repair. Multiple members of the CtBP-targeted gene network are selectively down-regulated in aggressive breast cancer subtypes. Differential expression of CtBP-targeted genes predicts poor clinical outcome in breast cancer patients, and elevated levels of CtBP in patient tumors predict shorter median survival. Finally, both CtBP promoter targeting and gene repression can be reversed by small molecule inhibition. These findings define broad roles for CtBP in breast cancer biology and suggest novel chromatin-based strategies for pharmacologic and metabolic intervention in cancer.

Expression of polycomb targets predicts breast cancer prognosis.

ABSTRACT Global changes in the epigenome are increasingly being appreciated as key events in cancer progression. The pathogenic role of enhancer of zeste homolog 2 (EZH2) has been connected to its histone 3 lysine 27 (H3K27) methyltransferase activity and gene repression; however, little is known about relationship of changes in expression of EZH2 target genes to cancer characteristics and patient prognosis. Here we show that through expression analysis of genomic regions with H3K27 trimethylation (H3K27me3) and EZH2 binding, breast cancer patients can be stratified into good and poor prognostic groups independent of known cancer gene signatures. The EZH2-bound regions were downregulated in tumors characterized by aggressive behavior, high expression of cell cycle genes, and low expression of developmental and cell adhesion genes. Depletion of EZH2 in breast cancer cells significantly increased expression of the top altered genes, decreased proliferation, and improved cell adhesion, indicating a critical role played by EZH2 in determining the cancer phenotype.

Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease

Pretransplant conditioning promotes minor mismatch GVHD by causing intestinal barrier loss and depleting NK cells. Double Trouble Before Transplant Graft-versus-host disease, a condition where transplanted immune cells attack the body of the transplant recipient, is a common complication of bone marrow or stem cell transplant. Although there are treatments available for treating graft-versus-host disease, they don’t always work well. Nalle et al. have now developed several different mouse models of this condition to provide insight into its causes. The authors used these mouse models to pinpoint two different ways that pretransplant conditioning contributes to graft-versus-host disease. This knowledge should help scientists search for new treatments against this disease, as well as identify safer ways to prepare patients for transplant. Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)–matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.

β-catenin regulates c-Myc and CDKN1A expression in breast cancer cells

We previously reported that the Wnt pathway is preferentially activated in basal‐like breast cancer. However, the mechanisms by which the Wnt pathway regulates down‐stream targets in basal‐like breast cancer, and the biological significance of this regulation, are poorly understood. In this study, we found that c‐Myc is highly expressed in the basal‐like subtype by microarray analyses and immunohistochemical staining. After silencing β‐catenin using siRNA, c‐Myc expression was decreased in non‐basal‐like breast cancer cells. In contrast, c‐Myc mRNA and protein expression were up‐regulated in the basal‐like breast cancer cell lines. Decreased c‐Myc promoter activity was observed after inhibiting β‐catenin by siRNA in non‐basal‐like breast cancer cells; however, inhibition of β‐catenin or over‐expression of dominant‐negative LEF1 had no effect on c‐Myc promoter activity in basal‐like breast cancer cell lines. In addition, CDKN1A mRNA and p21 protein expression were significantly increased in all breast cancer cell lines upon β‐catenin silencing. Interestingly, inhibiting β‐catenin expression alone did not induce apoptosis in breast cancer cell lines despite c‐Myc regulation, but we observed a modest increase of cells in the G1 phase of the cell cycle and decrease of cells in S phase upon β‐catenin silencing. Our findings suggest that the regulation of c‐Myc in breast cancer cells is dependent on the molecular subtype, and that β‐catenin‐mediated regulation of c‐Myc and p21 may control the balance of cell death and proliferation in breast cancer.

Apc Mutation Enhances PyMT-Induced Mammary Tumorigenesis

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, ApcMin/+ mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the ApcMin/+ mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the ApcMin/+ mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling.

The M2/Alternatively Activated Macrophage Phenotype Correlates with Aggressive Histopathologic Features and Poor Clinical Outcome in Early Stage Breast Cancer.

Background: Angiogenesis is critical for breast cancer progression. Within tumors, non-neoplastic cells assist tumor growth by producing growth factors and pro-angiogenic cytokines. Studies have demonstrated that tumor-associated macrophages (TAMs) are recruited to tumors before malignant conversion and are essential for promoting angiogenesis. We sought to study the role that macrophage phenotype—classically activated (M1) and alternatively activated (M2)—plays in the subsequent activation of the angiogenic pathway, with the goal of understanding the mechanisms underlying angiogenesis in the different molecular subtypes of breast cancer.Methods: 128 matching breast tumors, DCIS and normal tissues were obtained from the University of Chicago Breast Cancer SPORE tissue bank under IRB approved protocols. Tissue microarrays were constructed and molecular subtype was assigned based on immunohistochemical (IHC) staining into the following groups: luminal A (ER+, PR+, HER2-), luminal B (ER+, HER2+ or ER+, PR-), HER2-like (ER-, HER2+) and basal-like (ER-, HER2-, EGFR+ and/or CK5/6+). Macrophage phenotype was determined using double staining with CD68/CD163 (M2) and CD68/CD80 (M1). Microvessel density (MVD) was measured by IHC staining using anti-CD34. Staining quantification was performed independently by two pathologists. To control for intra-individual correlation, linear mixed-effects models were used to compare differences in % of M1 and M2 with disease progression. To evaluate the association of MVD with % of M1 and M2, bivariate plots were generated and Pearson9s correlation coefficients were calculated. Spearman9s correlation coefficients were used for the correlation between macrophage phenotype and tumor stage and grade. The Kaplan-Meier method was used to calculate overall survival.Results: Of the tumors studied, 88% were stage I-II. 17% were grade 1, 39% grade 2 and 44% grade 3. 70 were luminal A, 36 basal-like, 9 HER2-like and 6 luminal B. The ratio of M2:M1 increased with disease progression from normal breast to DCIS to invasive cancer (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 107.

Resistance to trastuzumab in HER2-positive mucinous invasive ductal breast carcinoma

Resistance to trastuzumab, either primary/de novo resistance or acquired/treatment-induced resistance, is a major clinical concern facing breast oncologists today.1 Here, we describe two cases of human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) with a mucin-producing component that were presumably resistant to trastuzumab.