EMT and the Warburg Effect in Breast Cancer
The Warburg effect (aerobic glycolysis) in cancer arises from the observation that tumor cells preferentially metabolize glucose to lactate through glycolysis, bypassing oxidative phosphorylation, even in the presence of oxygen. Although oncogene activation (e.g., Myc) can activate the Warburg effect via regulation of the catabolic pathway of glucose, the contribution of gluconeogenesis enzymes to this process is less clear. Fructose-1,6-bisphosphatase (FBP1) is a rate-limiting enzyme in gluconeogenesis. Dong and colleagues examined a role for FBP1 in the Warburg effect by studying the basal-like subtype of breast cancer, known to exhibit epithelial–mesenchymal transition (EMT), in part through activation of the Snail transcription factor. They first linked FBP1 and basal-like breast cancer by showing that FBP1 expression is inversely associated with Snail expression. FBP1 expression suppressed Snail-induced EMT and the basal-like phenotype in breast cancer cells. Snail overexpression in cell lines suppressed expression of FBP1, and chromatin immunoprecipitation revealed that the transcriptional repressor Snail was associated at the FBP1 promoter. Further work showed that Snail-mediated EMT resulted in changes in epigenetic marks at the FBP1 promoter, including increased G9a and H3K9me2 and decreased H3K9ac. Subsequently, FBP1 was shown to inhibit glucose uptake and activate oxidative phosphorylation. Mechanistically, the authors demonstrated that FBP1 suppressed pyruvate kinase (PKM2), a known activator of lactate production and the Warburg effect. Because FBP1 enhances oxygen consumption, they reasoned that it might stimulate activity of the mitochondrial electron transport system. Gene expression profiling showed that FBP1 overexpression resulted in upregulation of mitochondrial transcription factor B1M (TFB1M), a gene that is essential for mitochondrial biogenesis. Components of mitochondrial complex 1 were also upregulated as a result of FBP1 expression. This evidence suggests a direct role for FBP1 in tumorigenesis, as FBP1 overexpression inhibited tumor sphere formation, whereas FBP1 knockdown enhanced tumor sphere formation in breast cancer cells. This study implicates FBP1 as a potentially important link between Snail-mediated EMT and the Warburg effect in basal-like breast cancer.
miR-290, a Novel Suppressor of Breast Cancer Metastasis
Goldberger N, Walker RC, Kim CH, Winter S, Hunter KW. Inherited variation in miR-290 expression suppresses breast cancer progression by targeting the metastasis susceptibility gene Arid4b. Cancer Res 2013;73:2671–81.
Metastatic breast cancer remains a lethal disease with disappointing morbidity. An enhanced understanding of the mechanisms underlying disease progression could help to improve treatment options for these patients. Goldberger and colleagues identified microRNA-290 (miR-290) as a potential metastatic suppressor molecule in breast cancer while screening for factors involved in heritable onset of tumor latency and metastasis through a cross between highly metastatic FVB/N-TgN (MMTV-PyMT) and AKXD (AKR/J x DBA/2J) strains of mice. Highly metastatic mouse cell lines 6DT1 and Mtv-1 overexpressing miR-290 (pooled or single-cell clones) not only displayed a dramatic reduction of in vivo tumor growth but also showed reduced lung metastases when the cells were implanted orthotopically in the mammary fat pad of FVB/N mice. Using bioinformatics and functional studies, the authors identified a metastasis susceptibility gene, Arid4b (AT rich interactive domain 4B), as a direct target of miR-290. They observed a corresponding and noteworthy reduction in the mRNA and protein levels of Arid4b in cells overexpressing miR-290. Through microarray and quantitative real-time PCR analysis, specific upregulation of the estrogen receptor (ER) was detected in the miR-290–expressing tumors, and these results were positively correlated with significant elevation of apoptosis of these cells in vivo. In addition to changes in the ER signaling pathway, numerous other altered pathways, including pyrimidine and purine metabolism, aminoacyl-tRNA biosynthesis, cell-cycle control, and chromosomal replication, were identified. Thus, miR-290 appears to be a promising metastasis-suppressive molecule with potential applications for the development of novel strategies for breast cancer management. (Image from cited article courtesy of publisher.)
Alternative and Dangerous Routes to Invasive Carcinoma of the Skin
Cancer mortality is driven largely by the ability of tumor cells to metastasize to distant sites, yet our understanding of this process in vivo is limited. Skin carcinogenesis is typically thought to progress in a linear fashion from benign papillomas to invasive carcinomas, a subset of which undergo epithelial–mesenchymal transition (EMT) to spindle cell carcinomas. Wong and colleagues present data that challenge this view and suggest an alternative pathway characterized by reduced levels of H-Ras and EGFR signaling, decreased dependence on inflammation, and frequent deletion of the Ink4a/ARF (Cdkn2a) locus. The resulting tumor, referred to as class B carcinoma, in contrast to the class A carcinoma induced via the classical linear pathway, is highly invasive and has increased expression of markers of EMT and epithelial stem/progenitor cells. Using a two-stage skin carcinogenesis model, the authors observed that, whereas squamous cell carcinoma formation (class A tumors) increased in a linear manner with increased exposure to 12-O-tetradecanoylphorbol-13-acetate, the development of highly invasive spindle cell carcinomas with EMT properties (class B tumors) did not. These data suggest that class A and B tumors arise independently and may be dependent on different factors. Based on a combination of skin carcinogenesis models and mice with germline deletion of H-Ras, these data suggest that highly malignant class B tumors arise via an alternative pathway in which K-Ras mutations and deletion of Ink4a/ARF locus are more common and inflammation is less important. Future studies are needed to explore potential differences in the tumor cell of origin and determinants of tumor metastasis between class A and B skin carcinomas; however, the current data suggest important differences with therapeutic relevance.
BCL Family Members in Melanoma May Limit the Efficacy of BRAF-Directed Therapy
Haq R, Yokoyama S, Hawryluk EB, Jönsson GB, Frederick DT, McHenry K, et al. BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition. Proc Natl Acad Sci U S A 2013;110:4321–6.
Small-molecule inhibition of the mutated BRAF oncogene in patients with metastatic melanoma leads to variable and often transient objective responses. Although several mechanisms of acquired resistance have been discovered, far less is known about mechanisms that limit the primary efficacy of BRAF-directed therapy. Haq and colleagues conducted an integrated bioinformatic and functional analysis to identify genomically amplified therapeutic targets in several tumor types. This analysis led to the discovery of the antiapoptotic factor BCL2A1, located on chromosome 15q, as uniquely amplified in 30% to 40% of primary melanomas. The copy number status ofBCL2A1was correlated with mRNA/protein expression and inferior survival. BCL2A1, unlike other BCL2 family members, has not been previously described as a human oncogene and was shown here to be required for melanoma growth in knockdown and transformation assays. The expression of BCL2A1was restricted tomelanomas and was correlated with levels of MITF, a previously identified lineage-specific oncogene known to regulate melanoma growth and survival. Because amplification of BCL2 family members is postulated to limit the effectiveness of chemotherapy or targeted therapy, these authors also investigated whether BCL2A1 mediated intrinsic resistance to BRAF inhibitors. The novel combination of BRAF inhibitors and obatoclax (a pan-BCL2 family inhibitor) in BRAF-mutant model systems selectively enhanced apoptosis in vitro and markedly reduced tumor volumes in vivo. These findings suggest that BCL2A1 is a prognostic biomarker for poor outcome in patients with BRAF-mutant melanoma with the potential to be improved by concomitant treatment with a BCL2 antagonist.
Many studies have investigated the roles of human T cells and macrophages in the tumor microenvironment, but less is known about the immunoregulatory role of B cells in cancer, although results from mouse model studies suggest that both antibodies and B cell–derived cytokines can be tumor promoting. Karagiannis and colleagues hypothesized that a Th2-type inflammatory tumor microenvironment might favor alternatively activated humoral immunity with local expression of IgG4 antibodies in human melanoma. IgG4 is a relatively low abundant IgG subclass with weak complement and Fc receptor-binding activity. Its production is usually associated with prolonged exposure to antigen and is frequently observed in chronic inflammatory and autoimmune conditions. Increasing evidence suggests that IgG4 may induce clinical tolerance during allergen immunotherapy. In samples from patients with melanoma, the authors found elevated numbers of mature antibody-expressing B cells in primary and metastatic melanoma lesions. Furthermore, ex vivo stimulation of B cells enhanced the levels of key Th2-type cytokines, such as IL-4, IL-10, and VEGF, that could trigger IgG4 production. When compared with B cells from patient lymph nodes, blood, or healthy skin, tumor-associated B cells were polarized to produce IgG4. Also, serum IgG4 was inversely correlated with patient survival. In vitro, melanoma cells stimulated human B cells to produce the IgG4-polarizing cytokines and IgG4. The authors then engineered IgG1 and IgG4 antibodies of the same specificity against a melanoma-associated antigen, CSPG4. Unlike IgG1, IgG4 was unable to stimulate effector cell–mediated tumor killing In vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions through reduction of FcγRI activation. IgG4 also significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. These findings, in the context of melanoma, demonstrate a novel aspect of tumor induced immune escape in that B-cell infiltration and expression of IgG4 in the presence of IgG4-polarizing cytokines may promote an environment that counteracts antitumor immunity. These observations indicate that IgG4 may be elevated in other human cancers and could serve as a potential biomarker.
Stricker SH, Feber A, Engström PG, Carén H, Kurian KM, Takashima Y, et al. Widespread resetting of DNA methylation in glioblastoma initiating cells suppresses malignant cellular behavior in a lineage dependent manner. Genes Dev 2013;27:654–69.
It is increasingly clear that cancers show frequent epigenetic changes. However, the significance of these changes is often more difficult to ascertain. Stricker and colleagues reprogrammed glioblastoma multiforme (GBM) stem cells into human induced pluripotent stem (iPS) cells and then analyzed how the reprogramming of epigenetic marks influenced tumorigenicity. Starting with 14 GBM stem cell cultures, the authors were able to generate expandable iPS cell cultures in four of them, with complete reprogramming observed in two cell lines. Single nucleotide polymorphism array analysis showed one cell line to maintain cardinal genomic features of a proneural/classical lineage and the other to resemble a mesenchymal GBM. Although the two primary tumors showed characteristic GBM-specific DNA methylation abnormalities, the GBM-derived iPS cells showed erasure of many methylation marks and formed multilineage teratomas in vivo. One of the two iPS cell lines was subsequently differentiated toward a neural lineage. Orthotopic transplantation revealed infiltrating GBM-like tumors at a frequency similar to that of the parental GBM stem cell line. In contrast, differentiation of the iPS cell line toward a nonneural fate and subsequent injection into recipient mice led to benign noninfiltrating cartilaginous tumors. Thus, in the context of the neural lineage, resetting of GBM-associated epigenetic abnormalities was insufficient to override the cancer genome. In contrast, imposing an epigenome associated with an alternative developmental lineage more potently affected malignant behavior.
Note: Breaking Advances are written by Cancer Research Editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.