Gamze Karaca

Immunohistochemical and Clinical Characterization of the Basal-Like Subtype of Invasive Breast Carcinoma

Purpose: Expression profiling studies classified breast carcinomas into estrogen receptor (ER)+/luminal, normal breast-like, HER2 overexpressing, and basal-like groups, with the latter two associated with poor outcomes. Currently, there exist clinical assays that identify ER+/luminal and HER2-overexpressing tumors, and we sought to develop a clinical assay for breast basal-like tumors. Experimental Design: To identify an immunohistochemical profile for breast basal-like tumors, we collected a series of known basal-like tumors and tested them for protein patterns that are characteristic of this subtype. Next, we examined the significance of these protein patterns using tissue microarrays and evaluated the prognostic significance of these findings. Results: Using a panel of 21 basal-like tumors, which was determined using gene expression profiles, we saw that this subtype was typically immunohistochemically negative for estrogen receptor and HER2 but positive for basal cytokeratins, HER1, and/or c-KIT. Using breast carcinoma tissue microarrays representing 930 patients with 17.4-year mean follow-up, basal cytokeratin expression was associated with low disease-specific survival. HER1 expression was observed in 54% of cases positive for basal cytokeratins (versus 11% of negative cases) and was associated with poor survival independent of nodal status and size. c-KIT expression was more common in basal-like tumors than in other breast cancers but did not influence prognosis. Conclusions: A panel of four antibodies (ER, HER1, HER2, and cytokeratin 5/6) can accurately identify basal-like tumors using standard available clinical tools and shows high specificity. These studies show that many basal-like tumors express HER1, which suggests candidate drugs for evaluation in these patients.

Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma

Microarray profiling of invasive breast carcinomas has identified five distinct subtypes of tumors (luminal A, luminal B, normal breast-like, HER2 overexpressing, and basal-like) that are associated with different clinical outcomes. The basal-like subtype is associated with poor clinical outcomes and is the subtype observed in BRCA1-related breast cancers. The aim of this study was to characterize the histologic and immunophenotypic properties of breast basal-like carcinomas that were first positively identified using DNA microarray analysis. Detailed histologic review was performed on 56 tumors with known microarray profiles (23 basal-like, 23 luminal, and 12 HER2+). Immunohistochemistry for estrogen receptor (ER), HER2, EGFR, smooth muscle actin (SMA), p63, CD10, cytokeratin 5/6, cytokeratin 8/18, and vimentin was performed on 18 basal-like, 16 luminal, and 12 HER2+ tumors. The basal-like tumors were grade 3 ductal/NOS (21/23) or metaplastic (2/23) carcinomas that frequently showed geographic necrosis (17/23), a pushing border of invasion (14/23), and a stromal lymphocytic response (13/23). Most basal-like tumors showed immunoreactivity for vimentin (17/18), luminal cytokeratin 8/18 (15/18), EGFR (13/18), and cytokeratin 5/6 (11/18), while positivity for the myoepithelial markers SMA (4/18), p63 (4/18) and CD10 (2/18) was infrequent. All basal-like tumors tested were ER− and HER2−. Morphologic features significantly associated with the basal-like subtype included markedly elevated mitotic count (P<0.0001), geographic tumor necrosis (P=0.0003), pushing margin of invasion (P=0.0001), and stromal lymphocytic response (P=0.01). The most consistent immunophenotype seen in the basal-like tumors was negativity for ER and HER2, and positivity for vimentin, EGFR, cytokeratin 8/18, and cytokeratin 5/6. The infrequent expression of myoepithelial markers in basal-like carcinomas does not support a direct myoepithelial cell derivation of these tumors. These findings should further assist in the identification of basal-like carcinomas in clinical specimens, facilitating treatment and epidemiologic studies of this tumor subtype.

Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression.

The prognostication of head and neck squamous cell carcinoma (HNSCC) is largely based upon the tumor size and location and the presence of lymph node metastases. Here we show that gene expression patterns from 60 HNSCC samples assayed on cDNA microarrays allowed categorization of these tumors into four distinct subtypes. These subtypes showed statistically significant differences in recurrence-free survival and included a subtype with a possible EGFR-pathway signature, a mesenchymal-enriched subtype, a normal epithelium-like subtype, and a subtype with high levels of antioxidant enzymes. Supervised analyses to predict lymph node metastasis status were approximately 80% accurate when tumor subsite and pathological node status were considered simultaneously. This work represents an important step toward the identification of clinically significant biomarkers for HNSCC.

αB-Crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer

Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein alpha-basic-crystallin (alphaB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.

Mutation of GATA3 in human breast tumors

GATA3 is an essential transcription factor that was first identified as a regulator of immune cell function. In recent microarray analyses of human breast tumors, both normal breast luminal epithelium and estrogen receptor (ESR1)-positive tumors showed high expression of GATA3. We sequenced genomic DNA from 111 breast tumors and three breast-tumor-derived cell lines and identified somatic mutations of GATA3 in five tumors and the MCF-7 cell line. These mutations cluster in the vicinity of the highly conserved second zinc-finger that is required for DNA binding. In addition to these five, we identified using cDNA sequencing a unique mis-splicing variant that caused a frameshift mutation. One of the somatic mutations we identified was identical to a germline GATA3 mutation reported in two kindreds with HDR syndrome/OMIM #146255, which is an autosomal dominant syndrome caused by the haplo-insufficiency of GATA3. The ectopic expression of GATA3 in human 293T cells caused the induction of 73 genes including six cytokeratins, and inhibited cell line doubling times. These data suggest that GATA3 is involved in growth control and the maintenance of the differentiated state in epithelial cells, and that GATA3 variants may contribute to tumorigenesis in ESR1-positive breast tumors.

Hedgehog-Mediated Epithelial-to-Mesenchymal Transition and Fibrogenic Repair in Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Repair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors. METHODS Immature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc(+/-)) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD. RESULTS In cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc(+/-) mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD. CONCLUSIONS Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease

Liver inflammation is greater in nonalcoholic steatohepatitis (NASH) than steatosis, suggesting that immune responses contribute to nonalcoholic fatty liver disease (NAFLD) progression. Livers normally contain many natural killer T (NKT) cells that produce factors that modulate inflammatory and fibrogenic responses. Such cells are relatively depleted in steatosis, but their status in more advanced NAFLD is uncertain. We hypothesized that NKT cells accumulate and promote fibrosis progression in NASH. We aimed to determine if livers become enriched with NKT cells during NASH‐related fibrosis; identify responsible mechanisms; and assess if NKT cells stimulate fibrogenesis. NKT cells were analyzed in wildtype mice and Patched‐deficient (Ptc+/−) mice with an overly active Hedgehog (Hh) pathway, before and after feeding methionine choline‐deficient (MCD) diets to induce NASH‐related fibrosis. Effects of NKT cell‐derived factors on hepatic stellate cells (HSC) were examined and fibrogenesis was evaluated in CD1d‐deficient mice that lack NKT cells. NKT cells were quantified in human cirrhotic and nondiseased livers. During NASH‐related fibrogenesis in wildtype mice, Hh pathway activation occurred, leading to induction of factors that promoted NKT cell recruitment, retention, and viability, plus liver enrichment with NKT cells. Ptc+/− mice accumulated more NKT cells and developed worse liver fibrosis; CD1d‐deficient mice that lack NKT cells were protected from fibrosis. NKT cell‐conditioned medium stimulated HSC to become myofibroblastic. Liver explants were 2‐fold enriched with NKT cells in patients with non‐NASH cirrhosis, and 4‐fold enriched in patients with NASH cirrhosis. Conclusion: Hh pathway activation leads to hepatic enrichment with NKT cells that contribute to fibrosis progression in NASH. (HEPATOLOGY 2010;)

Smoothened is a master regulator of adult liver repair

When regenerative processes cannot keep pace with cell death, functional epithelia are replaced by scar. Scarring is characterized by both excessive accumulation of fibrous matrix and persistent outgrowth of cell types that accumulate transiently during successful wound healing, including myofibroblasts (MFs) and progenitors. This suggests that signaling that normally directs these cells to repair injured epithelia is deregulated. To evaluate this possibility, we examined liver repair during different types of liver injury after Smoothened (SMO), an obligate intermediate in the Hedgehog (Hh) signaling pathway, was conditionally deleted in cells expressing the MF-associated gene, αSMA. Surprisingly, blocking canonical Hh signaling in MFs not only inhibited liver fibrosis but also prevented accumulation of liver progenitors. Hh-sensitive, hepatic stellate cells (HSCs) were identified as the source of both MFs and progenitors by lineage-tracing studies in 3 other strains of mice, coupled with analysis of highly pure HSC preparations using flow cytometry, immunofluorescence confocal microscopy, RT-PCR, and in situ hybridization. The results identify SMO as a master regulator of hepatic epithelial regeneration based on its ability to promote mesenchymal-to-epithelial transitions in a subpopulation of HSC-derived MFs with features of multipotent progenitors.

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease

Objective Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. Design The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. Results When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18‒/‒ and CD1d‒/‒ mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. Conclusion Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.