Alexander C. Mackinnon

Expression and mutational analysis of MET in human solid cancers

MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) regulate a variety of cellular functions, many of which can be dysregulated in human cancers. Activated MET signaling can lead to cell motility and scattering, angiogenesis, proliferation, branching morphogenesis, invasion, and eventual metastasis. We performed systematic analysis of the expression of the MET receptor and its ligand HGF in tumor tissue microarrays (TMA) from human solid cancers. Standard immunohistochemistry (IHC) and a computerized automated scoring system were used. DNA sequencing for MET mutations in both nonkinase and kinase domains was also performed. MET was differentially overexpressed in human solid cancers. The ligand HGF was widely expressed in both tumors, primarily intratumoral, and nonmalignant tissues. The MET/HGF likely is functional and may be activated in autocrine fashion in vivo. MET and stem cell factor (SCF) were found to be positively stained in the bronchioalevolar junctions of lung tumors. A number of novel mutations of MET were identified, particularly in the extracellular semaphorin domain and the juxtamembrane domain. MET‐HGF pathway can be assayed in TMAs and is often overexpressed in a wide variety of human solid cancers. MET can be activated through overexpression, mutation, or autocrine signaling in malignant cells. Mutations in the nonkinase regions of MET might play an important role in tumorigenesis and tumor progression. MET would be an important therapeutic antitumor target to be inhibited, and in lung cancer, MET may represent a cancer early progenitor cell marker.

Mitochondria targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death

Cancer cells are long known to exhibit increased aerobic glycolysis, but glycolytic inhibition has not offered a viable chemotherapeutic strategy in part because of the systemic toxicity of antiglycolytic agents. However, recent studies suggest that a combined inhibition of glycolysis and mitochondrial function may help overcome this issue. In this study, we investigated the chemotherapeutic efficacies of mitochondria-targeted drugs (MTD) in combination with 2-deoxy-d-glucose (2-DG), a compound that inhibits glycolysis. Using the MTDs, termed Mito-CP and Mito-Q, we evaluated relative cytotoxic effects and mitochondrial bioenergetic changes in vitro. Interestingly, both Mito-CP and Mito-Q synergized with 2-DG to decrease ATP levels in two cell lines. However, with time, the cellular bioenergetic function and clonogenic survival were largely restored in some cells. In a xenograft model of human breast cancer, combined treatment of Mito-CP and 2-DG led to significant tumor regression in the absence of significant morphologic changes in kidney, liver, or heart. Collectively, our findings suggest that dual targeting of mitochondrial bioenergetic metabolism with MTDs and glycolytic inhibitors such as 2-DG may offer a promising chemotherapeutic strategy.

Transcription factor genes Smad4 and Gata4 cooperatively regulate cardiac valve development

We report that the dominant human missense mutations G303E and G296S in GATA4, a cardiac-specific transcription factor gene, cause atrioventricular septal defects and valve abnormalities by disrupting a signaling cascade involved in endocardial cushion development. These GATA4 missense mutations, but not a mutation causing secundum atrial septal defects (S52F), demonstrated impaired protein interactions with SMAD4, a transcription factor required for canonical bone morphogenetic protein/transforming growth factor-β (BMP/TGF-β) signaling. Gata4 and Smad4 genetically interact in vivo: atrioventricular septal defects result from endothelial-specific Gata4 and Smad4 compound haploinsufficiency. Endothelial-specific knockout of Smad4 caused an absence of valve-forming activity: Smad4-deficient endocardium was associated with acellular endocardial cushions, absent epithelial-to-mesenchymal transformation, reduced endocardial proliferation, and loss of Id2 expression in valve-forming regions. We show that Gata4 and Smad4 cooperatively activated the Id2 promoter, that human GATA4 mutations abrogated this activity, and that Id2 deficiency in mice could cause atrioventricular septal defects. We suggest that one determinant of the phenotypic spectrum caused by human GATA4 mutations is differential effects on GATA4/SMAD4 interactions required for endocardial cushion development.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

BackgroundRecent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells.MethodsIn this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed.ResultsBoth Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer.ConclusionsWe conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect.

Ets1 is required for proper migration and differentiation of the cardiac neural crest

Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1−/− mice have a nearly complete perinatal lethality. Histologic examination of Ets1−/− embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1−/− mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.

Depletion of M2-like tumor-associated macrophages delays cutaneous T-cell lymphoma development in vivo.

Macrophages have key roles in tumor development and invasion in several human cancers, but little is known about their pathogenic role in cutaneous T-cell lymphoma (CTCL). Herein, we used PCR arrays to profile the expression of inflammatory cytokines in 12 patients with mycosis fungoides (MF), the most common variant of CTCL. Compared with normal controls, MF skin displayed increased mRNA levels of macrophage-related cytokines. Moreover, we detected CD163, a reliable marker of tumor-associated macrophages, in the tumor microenvironment of MF biopsies. To demonstrate that macrophages had a role in CTCL tumorigenesis, we xenografted human CTCL tumor cells in immunocompromised mice and compared tumor development using clodronate-containing liposomes to deplete macrophages in mice. Mice treated with clodronate-containing liposomes show markedly less tumor growth compared with mice treated with phosphate-buffered saline-containing liposomes (P<0.001). We also noted a strong correlation between macrophage depletion and decreased expression of vascular marker, CD31, and lymphatic marker, podoplanin, suggesting a role for macrophages in angiogenesis. In vitro, clodronate-containing liposomes killed activated murine M2 macrophages, but not Hut78 cells, demonstrating selective ability to induce apoptosis in macrophages. Our data indicate that macrophages have a critical role in the progression of Hut78 cell tumor formation in skin, thus providing a new therapeutic strategy for CTCL.

Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome

Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Here, we developed a chromosome conformation capture (3C)-based multi-target sequencing technology and systematically examined three PC risk regions at the 8q24 locus and their potential regulatory targets across human genome in six cell lines. We observed frequent physical contacts of this risk locus with multiple genomic regions, in particular, inter-chromosomal interaction with CD96 at 3q13 and intra-chromosomal interaction with MYC at 8q24. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B and GSDMC and inter-chromosomal interaction gene CXorf36 in most of the six cell lines. Other gene regions appeared to be cell line-specific, such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in lymphoblastoid cell line. We further found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

Paxillin expression and amplification in early lung lesions of high-risk patients, lung adenocarcinoma and metastatic disease

Background Paxillin is a modular protein that localises to cell adhesion sites where it facilitates bidirectional communication between the intracellular actin cytoskeleton and the extracellular matrix. These complex and dynamic interactions are essential for cell adhesion, cell migration and cell survival. The authors have previously demonstrated that paxillin is overexpressed in lung cancer tissues and identified somatic paxillin mutations in 9% of lung cancers. A murine in vivo xenograft model of the most common paxillin mutation (A127T) showed increased cell proliferation and invasive tumour growth, establishing an important role for paxillin in the development of lung cancer. Methods The authors analysed 279 bronchoscopy-aided biopsy specimens from 92 high-risk patients. Adenocarcinoma with bronchioloalveolar features and pure bronchioloalveolar carcinoma (BAC) were analysed with fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC). Results Paxillin is overexpressed in premalignant areas of hyperplasia, squamous metaplasia and goblet cell metaplasia, as well as dysplastic lesions and carcinoma in high-risk patients. Concordance between increased paxillin gene copy number and paxillin overexpression was observed in cases of adenocarcinoma eusomic for chromosome 12. Conclusions Paxillin overexpression occurs during the earliest stages of lung cancer development. FISH and IHC analysis of lung adenocarcinoma suggests that relatively small-scale genomic rearrangements of chromosome 12 are associated with paxillin overexpression in lung adenocarcinoma.

Identification of histologically distinct conventional adenomas that arise predominately in patients with sessile serrated adenomas.

We have recently shown that a study population of patients with at least 1 sessile serrated adenoma (SSA) are 4 times more likely to harbor synchronous serrated polyps [SSAs, traditional serrated adenomas (TSAs) and right sided hyperplastic polyps] than a unselected population of patients. However, 35% of the polyps in the study patients were conventional adenomas (CAds). We hypothesized that the CAds in these study patients would have histologic and molecular differences compared with CAds from a control population without sessile serrated adenomas. To this end, 104 study and 79 control CAds were analyzed according to 9 histologic criteria. A subset of these polyps was also screened for BRAF mutations, KRAS mutations, CpG island methylation, and MUC6 expression. A total of 31 study CAds and 2 control CAds had atypical histologic features (bright cytoplasmic eosinophilia ± focal serrations and crypt dilatation). None of the adenomas tested had mutations in BRAF or KRAS. Evidence of low levels of CpG island methylation was seen in 35% of the atypical CAds and in only 4.5% of the typical CAds. In addition, these atypical CAds were more likely to express MUC6. Thus, the presence of cytoplasmic eosinophilia with or without focal serrations and crypt dilatation identifies a subset of CAds with characteristics of the serrated neoplasia pathway. These atypical CAds occur more commonly in patients predisposed to developing SSAs and suggest the presence of a mucosal field defect in these patients.

Role of immunohistochemistry in the diagnosis of sebaceous carcinoma: a clinicopathologic and immunohistochemical study.

Abstract:Sebaceous carcinoma (SC) is a relatively uncommon malignant epithelial neoplasm with a predilection for the periocular region. The diagnosis of SC can be difficult to make at initial presentation, as it can clinically and histopathologically resemble other common benign and malignant epithelial lesions. A diagnosis of SC is made by confirmation of sebaceous differentiation of neoplastic cells, which can often be accomplished by conventional microscopic findings; however, its recognition may be sometimes difficult and requires ancillary studies such as immunohistochemistry (IHC). Many studies have evaluated the role of IHC as a potential technique to differentiate SC from its mimics; however, most of these studies have used a limited panel of antibodies with variable results. The aim of this study was to determine the efficacy of IHC in the diagnosis of SC and to provide some guidelines for interpretation in the diagnosis of these neoplasms. We studied 27 cases of SC with a broad panel of IHC markers using a tissue microarray technique. We also studied 21 control cases of basal cell carcinoma (BCC) and 22 control cases of squamous cell carcinoma (SCC). Representative tissue cores were taken and processed from each case, and the tissue microarrays were stained by standard methods using antibodies to EMA, CK7, Ber-EP4, Factor XIIIA, androgen receptor, p53, adipophilin, progesterone receptor membrane component 1 (PGRMC1), squalene synthase (SQS), and alpha/beta hydrolase domain-containing protein 5 (ABHD5). Our studies show that EMA was expressed in all cases of SC, CK7 was expressed in 24 of 27 cases, Ber-EP4 was expressed in 7 of 27 cases, Factor XIIIA was negative in all cases, androgen receptor was expressed in 9 of 27 cases, P53 was expressed in 12 of 27 cases, adipophilin was expressed in all cases, PGRMC1 was expressed in 22 of 27 cases, SQS was expressed in 11 of 27 cases, and ABHD5 was expressed in 9 of 27 cases. EMA was negative in all cases of BCC, CK7 was expressed in 6 of 21 cases, Ber-EP4 was expressed in 21 of 21 cases, Factor XIIIA was negative in all cases, androgen receptor was expressed in 3 of 21 cases, P53 was expressed in 4 of 21 cases, adipophilin, PGRMC1, SQS, and ABHD5 were negative in all cases of BCC. Similarly, EMA was expressed in 16 of 22 cases of SCC, CK7 was expressed in 2 of 22 cases, Ber-EP4, Factor XIIIA, and androgen receptor were negative in all cases, P53 was expressed in 3 of 22 cases, adipophilin, PGRMC1, SQS, and ABHD5 were negative in all cases of SCC. Our study indicates that adipophilin represents a sensitive and reliable marker for the diagnosis of SC and can be of help in separating this tumor from some of its mimics. Additionally, inclusion of various epithelial markers in the panel will be of help if adequately used. Other antibodies against the PAT family of lipid droplet-associated proteins including PGRMC1, SQS, and ABHD5 were not as sensitive as adipophilin for identifying sebaceous differentiation and may therefore not be as useful for differential diagnosis as adipophilin.