Ramsi Haddad

Molecular profiling of human hepatocellular carcinoma defines mutually exclusive interferon regulation and insulin-like growth factor II overexpression.

Molecular subtyping of human hepatocellular carcinoma (HCC) with potential mechanistic and therapeutic impact has not been achieved thus far. We have analyzed the mRNA expression patterns of 43 different human HCC samples and 3 HCC cell lines in comparison with normal adult liver using high-density cDNA microarrays. Two main groups of HCC, designated group A (65%) and group B (35%), were distinguished based on clustering of the most highly varying genes. Group A HCCs were characterized by induction of a number of interferon (IFN)-regulated genes, whereas group B was characterized mainly by down-regulation of several apoptosis-relevant and IFN-regulated genes. The number of apoptotic tumor cells and tumor-infiltrating lymphocytes was significantly higher in tumors of group A as compared with those of group B. Based on the expression pattern, group B was further subdivided into two subgroups, designated subgroup B1 (6 of 43 tumors, 14%) and subgroup B2 (9 of 43 tumors, 21%). A prominent characteristic of subgroup B1 was high overexpression of insulin-like growth factor (IGF)-II. All tested HCC cell lines expressed equally high concentrations of IGF-II transcripts and co-segregated with group B1 in clustering. IGF-II overexpression and induction of IFN-related genes were mutually exclusive, even when analysis was extended to other cancer expression profile studies. Moreover, IFN-γ treatment substantially reduced IGF-II expression in HCC cells. In conclusion, cDNA microarray analyses provided subtyping of HCCs that is related to intratumor inflammation and tumor cell apoptosis. This profiling may be of mechanistic and therapeutic impact because IGF-II overexpression has been linked to reduced apoptosis and increased proliferation and may be accessible to therapeutic intervention.

EGFR/Met association regulates EGFR TKI resistance in breast cancer

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.

Frequency and type of epidermal growth factor receptor mutations in African Americans with non-small cell lung cancer

Background: Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict response to tyrosine kinase inhibitors. Mutations occur more commonly in never smokers and East Asians, but there are conflicting reports on the frequency of EGFR mutations in tumors from African Americans. Methods: Tumors from 67 African American and 77 white participants in previous case-control studies of lung cancer were selected to determine EGFR mutational status. Mutation analysis was performed using the Sequenom mass array analyzer (Sequenom, San Diego, CA). Results: Overall, 13.9% of the study population carried an EGFR mutation. EGFR mutations occurred in 11.9% of tumors from African Americans compared with 15.6% in whites (p = 0.53). All mutations found in African Americans were deletions in exon 19. The majority of mutations were found in nonsmokers among both African Americans (7/8) and whites (8/12). Conclusion: These results indicate that African Americans with NSCLC harbor somatic EGFR mutations at a frequency similar to whites with NSCLC. Thus, clinicians should not use race as a clinical decision parameter for the use of EGFR-tyrosine kinase inhibitors.

Methylation-associated silencing of SFRP1 with an 8p11-12 amplification inhibits canonical and non-canonical WNT pathways in breast cancers

Recently, we analysed the 8p11‐12 genomic region for copy number and gene expression changes in a panel of human breast cancer cell lines and primary specimens. We found that SFRP1 (Secreted frizzled related protein 1) is frequently under expressed even in breast tumours with copy number increases in this genomic region. SFRP1 encodes a WNT signalling antagonist, and plays a role in the development of multiple solid tumour types. In this study, we analysed methylation‐associated silencing of the SFRP1 gene in breast cancer cells with the 8p11‐12 amplicon, and investigated the tumour suppressor properties of SFRP1 in breast cancer cells. SFRP1 expression was markedly reduced in both the breast cancer cell lines and primary tumour specimens relative to normal primary human mammary epithelial cells even when SFRP1 is amplified. Suppression of SFRP1 expression in breast cancer cells with an SFRP1 gene amplification is associated with SFRP1 promoter methylation. Furthermore, restoration of SFRP1 expression suppressed the growth of breast cancer cells in monolayer, and inhibited anchorage independent growth. We also examined the relationship between the silencing of SFRP1 gene and WNT signalling in breast cancer. Ectopic SFRP1 expression in breast cancer cells suppressed both canonical and non‐canonical WNT signalling pathways, and SFRP1 expression was negatively associated with the expression of a subset of WNT responsive genes including RET and MSX2. Thus, down‐regulation of SFRP1 can be triggered by epigenetic and/or genetic events and may contribute to the tumourigenesis of human breast cancer through both canonical and non‐canonical WNT signalling pathways.

Transforming properties of TC-1 in human breast cancer: Interaction with FGFR2 and β-catenin signaling pathways

Breast cancer development is associated with gene amplification and over expression that is believed to have a causative role in oncogenesis. Previous studies have demonstrated that over expression of TC‐1(C8orf4) mRNA occurs in ∼50% of breast cancer cell lines and primary tumor specimens. Here, we show that TC‐1 has transforming properties in human mammary epithelial (HME) cells and its expression is mechanistically linked to FGFR signaling cascades. In vitro experiments demonstrate that TC‐1 over expression mediates both anchorage‐independent and growth factor‐independent proliferation of HME cells. TC‐1 was down regulated by the FGFR inhibitor PD173074 in the breast cancer cell line SUM‐52 that also has an FGFR2 gene amplification and over expression. Furthermore, forced expression of FGFR2 in HME cells increased the level of expression of endogenous TC‐1 mRNA. TC‐1 has been implicated as a modulator of Wnt/β‐catenin signaling in 293 cells and in gastric cancer cells. However, while we did find increased expression of a subset of β‐catenin target genes in TC‐1 over expressing cells, we did not find an association of TC‐1 with global expression of β‐catenin target genes in our cells. Taken together, our data suggest that TC‐1 over expression is transforming and may link with the FGFR pathway in a subset of breast cancer.

Two members of the TRiC chaperonin complex, CCT2 and TCP1 are essential for survival of breast cancer cells and are linked to driving oncogenes.

Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer.

HER-2 Signaling, Acquisition of Growth Factor Independence, and Regulation of Biological Networks Associated with Cell Transformation

Activated oncogenes are the dominant drivers of malignant progression in human cancer, yet little is known about how the transformation from proto-oncogene to activated oncogene drives the expression of transformed phenotypes. An isogenic model of HER-2-mediated transformation of human mammary epithelial cells was used along with HER-2-amplified human breast cancers to investigate how HER-2 activation alters its properties as a signaling molecule and changes the networks of HER-2-regulated genes. Our results show that full oncogenic activation of HER-2 is the result of a transition in which activated HER-2 acquires dominant signaling properties that qualitatively alter the network of genes regulated by the activated oncogene compared with the proto-oncogene. Consequently, gene expression programs related to invasion, cell stress, and stemness become regulated by HER-2 in a manner not observed in nontransformed cells, even when HER-2 is overexpressed. Our results offer novel insights into biological processes that come under the control of HER-2 after it acquires full oncogenic potential.

Racial differences in oncogene mutations detected in early stage, low grade endometrial cancers

Objective To describe the pattern and frequency of oncogene mutations in white and African American women with endometrial cancer and to determine if racial differences in oncogene mutations exist among women with pathologically similar tumors. Methods Patients with endometrial cancer from a large urban hospital were identified through medical records, and representative formalin-fixed paraffin-embedded tumor blocks were retrieved. The study sample included 150 patients (84 African Americans) who underwent total abdominal hysterectomy for endometrial cancer. The Sequenom MassARRAY system and the OncoCarta Assay version 1.0 (Sequenom) were used to test for 238 mutations in 19 common oncogenes. The χ2 test and the Fisher exact test were used to assess differences in distribution of variables by race and oncogene mutation status. Results There were 20 mutations identified in 2 oncogenes (PIK3CA and KRAS) in tumors from 19 women (12.7%). Most of the mutations were found in PIK3CA (16/20). Thirteen percent of endometrioid tumors harbored mutations (11 PIK3CA and 2 KRAS) as did 29% of the malignant mixed Mullerian tumors (3 PIK3CA and 1 KRAS). There were no observed mutations in serous, clear cell, or mucinous tumor types. Among low-grade endometrioid cancers, tumors from African American patients were significantly associated with harboring either a KRAS or PIK3CA mutation (P = 0.04), with 7 PIK3CA mutations and all 4 KRAS mutations identified in African American women. Conclusions This study provides preliminary evidence that oncogene mutation frequency of some subtypes of histologically similar endometrial carcinoma differ by race. Additional studies are needed to further explore this phenomenon in patients with endometrial carcinoma.

Oncogene Mutation Survey in MPNST Cell Lines Enhances the Dominant Role of Hyperactive Ras in NF1 Associated Pro-Survival and Malignancy

Malignant peripheral nerve sheath tumors (MPNST) are a type of soft tissue sarcoma that can be associated with germline mutations in Neurofibromatosis type 1 (NF1) or may occur sporadically. Although the etiology of MPNST is poorly understood, it is clear that a loss of function of the NF1 gene, encoding a Ras-GAP, is an important factor in the tumorigenesis of the inherited form of MPNST. Tumor latency in NF1 patients suggests that additional mutational events are probably required for malignancy. In order to define oncogene mutations associated with 5 MPNST cell lines, we assayed the 238 most frequent mutations in 19 commonly activated oncogenes using mass spectroscopy-based analysis. All 238 mutation sites in the assayed oncogenes were determined to harbor only wild-type sequences. These data suggest that hyperactive Ras resulting from the loss function of neurofibromin may be sufficient to set up the direction of malignant transformation of Schwann cells to MPNST.

Constitutive c-Met phosphorylation mediates growth in the absence of EGFR kinase activity in breast cancer cells dependent on EGFR expression for growth.

Abstract #4059 Breast cancers are not responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) even though 30% of breast cancers overexpress the EGFR. We have observed that EGFR remains tyrosine phosphorylated in breast cancer cells that do not require EGFR kinase activity for growth, yet express high levels of EGFR. In one such cell line, SUM229, we found that this EGFR kinase-independent EGFR tyrosine phosphorylation encompasses all of the tyrosine phosphorylation sites of the EGFR for which antibodies are available, with the exception of tyrosine 1148. Using shRNA lentiviral constructs targeting EGFR, we also found that EGFR expression is required for the growth of the SUM229 cells, even though the kinase activity is not required. In addition, we found that this aberrant tyrosine phosphorylation and growth in the absence of EGFR kinase activity is mediated in part by c-Src and c-Met kinase activity. Interestingly, c-Met is constitutively phosphorylated in the SUM229 cells. This study describes the mechanism of c-Met constitutive activity and defines the Met-mediated signaling pathways to growth and survival in the absence of EGFR kinase activity. Specifically, through sequence analysis of mRNA from SUM229 cells, c-Met is not mutated, confirming the lack of identification of c-Met mutations in breast cancer. In addition, using publically available databases and our own immunoblotting experiments, c-Met is neither amplified nor overexpressed at the mRNA or protein level in the SUM229 cells. Similarly, ligand-mediated regulators of c-Met phosphorylation, including HGF, HGFAC, HAI-1, and HA1-2 are not overexpressed at the mRNA level and HGF protein expression and secretion are comparable to levels seen in breast cancer cells without c-Met constitutive phosphorylation. However, c-Met localization to the cell surface appears to be elevated in the SUM229 cells and c-Met co-immunoprecipitates with EGFR under these conditions. Taken together, these data suggest that c-Met and EGFR association at the cell surface may be mediating c-Met constitutive phosphorylation in the absence of EGFR kinase activity. With respect to the signaling pathways that mediate EGFR kinase-independent, yet c-Met kinase-dependent growth in SUM229 cells we have found that the phosphorylation of Akt, MAPK, p38MAPK, and JNK1/2 are unaffected by inhibition of both EGFR and c-Met kinase activities. Interestingly, the phosphorylation of Jak2, PLCg, and Gab1 are decreased when EGFR and c-Met kinases are inhibited. These data suggest that pathways signaling through Jak2, PLCg, and/or Gab1 may be mediating growth in the absence of EGFR kinase activity. In preliminary data, we know that these pathways do not involve PKC, Akt, or PI3-Kinase. Therefore, current investigations are focused on the STAT family of proteins as well as the Rho/Rac/Cdc42 mediated signaling pathways. Taken together, these data suggest that c-Met mediates a unique signaling pathway in SUM229 cells that stimulates both growth and survival in the absence of EGFR kinase activity. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4059.