Andrey Khramtsov

Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors

BackgroundAlthough numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors.ResultsUnsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of histologies and expression profiles developed. In many models, similarities to human breast tumors were recognized, including proliferation and human breast tumor subtype signatures. Significantly, tumors of several models displayed characteristics of human basal-like breast tumors, including two models with induced Brca1 deficiencies. Tumors of other murine models shared features and trended towards significance of gene enrichment with human luminal tumors; however, these murine tumors lacked expression of estrogen receptor (ER) and ER-regulated genes. TgMMTV-Neu tumors did not have a significant gene overlap with the human HER2+/ER- subtype and were more similar to human luminal tumors.ConclusionMany of the defining characteristics of human subtypes were conserved among the mouse models. Although no single mouse model recapitulated all the expression features of a given human subtype, these shared expression features provide a common framework for an improved integration of murine mammary tumor models with human breast tumors.

Population Differences in Breast Cancer: Survey in Indigenous African Women Reveals Over-Representation of Triple-Negative Breast Cancer

PURPOSE Compared with white women, black women experience a disproportionate burden of aggressive breast cancer for reasons that remain unknown and understudied. In the first study of its kind, we determined the distribution of molecular subtypes of invasive breast tumors in indigenous black women in West Africa. PATIENTS AND METHODS The study comprised 507 patients diagnosed with breast cancer between 1996 and 2007 at six geographic regions in Nigeria and Senegal. Formalin-fixed and paraffin-embedded sections were constructed into tissue microarrays and immunostained with 15 antibodies. Five molecular subtypes were determined, and hierarchical cluster analysis was conducted to explore subgroups for unclassified cases. RESULTS The mean (+/- standard deviation) age of 378 patients in the first cohort was 44.8 +/- 11.8 years, with the majority of women presenting with large (4.4 +/- 2.0 cm) high-grade tumors (83%) in advanced stages (72% node positive). The proportions of estrogen receptor (ER) -positive, progesterone receptor-positive, and human epidermal growth factor receptor 2 (HER2) -positive tumors were 24%, 20%, and 17%, respectively. Triple negativity for these markers was predominant, including basal-like (27%) and unclassified subtype (28%). Other subtypes were luminal A (27%), luminal B (2%), and HER2 positive/ER negative (15%). The findings were replicated in the second cohort of 129 patients. The unclassified cases could be grouped into a bad prognosis branch, with expression of vascular endothelial growth factor, B-cell lymphoma extra-large protein, and Cyclin E, and a good prognosis branch, with expression of B-cell lymphoma protein 2 and Cyclin D1. CONCLUSION These findings underscore the urgent need for research into the etiology and treatment of the aggressive molecular subtypes that disproportionately affect young women in the African diaspora.

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.

Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression.

We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor‐binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERα and c‐MYC gene targets were knocked down to identify mediators of E2‐stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2‐inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers.

A Selective Small Molecule Inhibitor of c-Met, PHA665752, Inhibits Tumorigenicity and Angiogenesis in Mouse Lung Cancer Xenografts

The c-Met receptor tyrosine kinase is emerging as a novel target in many solid tumors, including lung cancer. PHA-665752 was identified as a small molecule, ATP competitive inhibitor of the catalytic activity of the c-Met kinase. Here, we show that treatment with PHA665752 reduced NCI-H69 (small cell lung cancer) and NCI-H441 (non-small cell lung cancer) tumorigenicity in mouse xenografts by 99% and 75%, respectively. Reduction in tumor size was also observed by magnetic resonance imaging of tumors in mice. PHA665752 inhibited c-Met phosphorylation at the autophosphorylation and c-Cbl binding sites in mouse xenografts derived from non-small cell lung cancer cell lines (NCI-H441 and A549) and small cell lung cancer cell line (NCI-H69). PHA665752 also inhibited angiogenesis by >85% in all the abovementioned cell lines and caused an angiogenic switch which resulted in a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These studies show the feasibility of selectively targeting c-Met with ATP competitive small molecule inhibitors and suggest that PHA665752 may provide a novel therapeutic approach to lung cancer.

Prognostic Significance of E-Cadherin Protein Expression in Pathological Stage I-III Endometrial Cancer

Purpose: Decreased expression of E-cadherin in endometrial cancer cells is associated with adverse prognostic features. This study aimed to evaluate the prognostic significance of decreased E-cadherin expression in patients with endometrial cancer. Experimental Design: Between 1992 and 1999, 102 endometrial cancer patients with stage I-III disease underwent primary surgery at the University of Chicago. Representative tissue specimens were immunostained with a monoclonal antibody to E-cadherin. A semiquantitative evaluation scale was developed based on the percentage of endometrial cancer cells with membranous E-cadherin staining. Tissue sections were scored as “3” if >75%, “2” if 25–75%, “1” if 5–25%, and “0” if <5% of cells stained. E-Cadherin staining was correlated with overall survival (OS), cause-specific survival (CSS), progression-free survival (PFS), and extrapelvic progression. Multivariate Cox proportional hazards modeling was used to estimate hazard ratios, controlling for clinicopathological characteristics and adjuvant treatment. Median follow-up for the study group was 58.5 months. Results: E-Cadherin staining was scored as 0, 1, 2, and 3 in 29.4%, 18.6%, 26.5%, 25.5% of cases, respectively. E-Cadherin expression was positively correlated with myometrial invasion (Kendall τ: 0.30, P < 0.01), and negatively correlated with grade (Kendall τ: −0.13, P = 0.15) and papillary serous or clear cell histology (Kendall τ: −0.14, P = 0.12). Five-year actuarial OS, CSS, PFS, and extrapelvic recurrence rates for negative (score = 0), heterogeneous (score = 1–2), and positive (score = 3) staining were as follows: OS, 69.2 versus 75.7 versus 81.0% (P = 0.64); CSS, 78.8 versus 91.2 versus 95.5% (P = 0.19); PFS, 69.1 versus 88.6 versus 92.2% (P = 0.079), and extrapelvic progression, 20.8 versus 7.3 versus 4.0% (P = 0.17). On multivariate Cox regression, a higher E-cadherin expression score was associated with decreased overall mortality [hazard ratio (HR), 0.59; 95% confidence interval (CI), 0.34–1.03; P = 0.066), and statistically significant decreases in endometrial cancer mortality (HR, 0.23; 95% CI, 0.055–0.94; P = 0.040), disease progression (HR, 0.28; 95% CI, 0.10–0.77; P = 0.014), and extrapelvic recurrence (HR, 0.24; 95% CI, 0.062–0.97; P = 0.045). Conclusions: Decreased E-cadherin expression is an independent prognostic factor for disease progression and mortality in pathological stage I-III endometrial cancer. Evaluation of E-cadherin expression may aid in the selection of patients for more aggressive adjuvant therapy.

Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo

Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting.

Molecular Analysis of Colorectal Tumors within a Diverse Patient Cohort at a Single Institution

Purpose: African American colorectal cancer patients have worse survival outcomes than Caucasian patients. To determine whether differences exist in the molecular mechanisms driving colorectal cancer between African Americans and Caucasians, we characterized patient tumors from a single institution by assessing genetic alterations involved in colorectal cancer progression and response to treatment. Experimental Design: We retrospectively examined 448 African Americans and Caucasians diagnosed with colorectal cancer at The University of Chicago Medical Center between 1992 and 2002. Microsatellite instability (MSI) status was determined by genotyping the BAT25, BAT26, BAT40, D5S346, and BAX loci. Mutations in KRAS codons 12 and 13 and BRAF codon 600 were identified by direct sequencing. MSI and detected mutations were correlated with clinicopathologic features. Results: Overall, no difference existed in MSI or BRAF mutation frequencies between African Americans and Caucasians. However, African Americans with microsatellite stable (MSS)/MSI-low (MSI-L) tumors had a higher proportion of KRAS mutations than Caucasians (34% vs. 23%, P = 0.048) that was isolated to proximal colon cancers and primarily driven by mutations in codon 13. There was no racial difference in receipt of chemotherapy, but African Americans with MSS/MSI-L tumors had a 73% increased risk of death over Caucasians that could not be explained by known prognostic factors. Conclusions: The significantly higher risk of death among African Americans with MSS/MSI-L tumors may be related to differences in the distribution of factors influencing response to standard therapies. These data underscore the need for further research into the molecular mechanisms driving colorectal cancer progression in underserved and understudied populations. Clin Cancer Res; 18(2); 350–9. ©2011 AACR.

Enhanced transduction of malignant glioma with a double targeted Ad5/3-RGD fiber-modified adenovirus

Malignant brain tumors remain refractory to adenovirus type 5 (Ad5)–based gene therapy, mostly due to the lack of the primary Ad5 receptor, the coxsackie and adenovirus receptor, on brain tumor cells. To bypass the dependence on coxsackie and adenovirus receptor for adenoviral entry and infectivity, we used a novel, double targeted Ad5 backbone–based vector carrying a chimeric Ad5/3 fiber with integrin-binding RGD motif incorporated in its Ad3 knob domain. We then tested the new virus in vitro and in vivo in the setting of malignant glioma. Ad5/3-RGD showed a 10-fold increase in gene expression in passaged cell lines and up to 75-fold increase in primary tumors obtained from patients relative to the control. These results were further corroborated in our in vivo human glioma xenograft model, where the Ad5/3-RGD vector showed a 1,000-fold increase in infectivity as compared with the control. Taken together, our findings indicate that Ad5/3-RGD may be a superior vector for applications in glioma gene therapy and therefore warrants further attention in the field of neuro-oncology. [Mol Cancer Ther 2006;5(9):2408–17]

Concordance in Histological and Biological Parameters Between First and Second Primary Breast Cancers

Women with breast cancer are more likely to have a second breast cancer than women in the general population are to have a primary cancer. However, the biological relationship between primary and second breast cancers is not clear.