Identification of Two Genetic Loci Associated with Leukopenia after Chemotherapy in Patients with Breast Cancer.

PURPOSE: To identify molecular predictors of grade 3/4 neutropenic or leukopenic events (NLE) after chemotherapy using a genome-wide association study (GWAS). EXPERIMENTAL DESIGN: A GWAS was performed on patients in the phase III chemotherapy study SUCCESS-A (n = 3,322). Genotyping was done using the Illumina HumanOmniExpress-12v1 array. Findings were functionally validated with cell culture models and the genotypes and gene expression of possible causative genes were correlated with clinical treatment response and prognostic outcomes. RESULTS: One locus on chromosome 16 (rs4784750; NLRC5; P = 1.56E-8) and another locus on chromosome 13 (rs16972207; TNFSF13B; P = 3.42E-8) were identified at a genome-wide significance level. Functional validation revealed that expression of these two genes is altered by genotype-dependent and chemotherapy-dependent activity of two transcription factors. Genotypes also showed an association with disease-free survival in patients with an NLE. CONCLUSIONS: Two loci in NLRC5 and TNFSF13B are associated with NLEs. The involvement of the MHC I regulator NLRC5 implies the possible involvement of immuno-oncological pathways.

Transdifferentiation of glioblastoma stem-like cells into mural cells drives vasculogenic mimicry in glioblastomas.

Recent evidence has shown that glioblastoma stem-like cells (GSCs) can transdifferentiate into endothelial cells and vascular-like tumor cells. The latter pattern of vascularization indicates an alternative microvascular circulation known as vasculogenic mimicry (VM). However, it remains to be clarified how the GSC-driven VM makes a significant contribution to tumor vasculature. Here, we investigated 11 cases of glioblastomas and found that most of them consisted of blood-perfused vascular channels that coexpress mural cell markers smooth muscle α-actin and platelet-derived growth factor receptor ß, epidermal growth factor receptor, and vascular endothelial growth factor receptor 2 (Flk-1), but not CD31 or VE-cadherin. This microvasculature coexisted with endothelial cell-associated vessels. GSCs derived from patients with glioblastomas developed vigorous mural cell-associated vascular channels but few endothelial cell vessels in orthotopic animal models. Suppression of Flk-1 activity and gene expression abrogated GSC transdifferentiation and vascularization in vitro, and inhibited VM in animal models. This study establishes mural-like tumor cells differentiated from GSCs as a significant contributor to microvasculature of glioblastoma and points to Flk-1 as a potential target for therapeutic intervention that could complement current anti-angiogenic treatment.

Glioblastoma-derived tumor cells induce vasculogenic mimicry through Flk-1 protein activation.

Glioblastoma (GBM) is extremely aggressive and essentially incurable. Its malignancy is characterized by vigorous microvascular proliferations. Recent evidence has shown that tumor cells display the ability to drive blood-perfused vasculogenic mimicry (VM), an alternative microvascular circulation independent of endothelial cell angiogenesis. However, molecular mechanisms underlying this vascular pathogenesis are poorly understood. Here, we found that vascular channels of VM in GBM were composed of mural-like tumor cells that strongly express VEGF receptor 2 (Flk-1). To explore a potential role of Flk-1 in the vasculogenesis, we investigated two glioblastoma cell lines U87 and GSDC, both of which express Flk-1 and exhibit a vascular phenotype on Matrigel. Treatment of both cell lines with either Flk-1 gene knockdown or Flk-1 kinase inhibitor SU1498 abrogated Flk-1 activity and impaired vascular function. Furthermore, inhibition of Flk-1 activity suppressed intracellular signaling cascades, including focal adhesion kinase and mitogen-activated protein kinase ERK1/2. In contrast, blockade of VEGF activity by the neutralizing antibody Bevacizumab failed to recapitulate the impact of SU1498, suggesting that Flk-1-mediated VM is independent of VEGF. Xenotransplantation of SCID/Beige mice with U87 cells and GSDCs gave rise to tumors harboring robust mural cell-associated vascular channels. Flk-1 shRNA restrained VM in tumors and subsequently inhibited tumor development. Collectively, all the data demonstrate a central role of Flk-1 in the formation of VM in GBM. This study has shed light on molecular mechanisms mediating tumor aggressiveness and also provided a therapeutic target for patient treatment.

The novel lupus antigen related protein acheron enhances the development of human breast cancer.

Acheron (Achn) is a new member of the Lupus antigen family of RNA binding proteins. Previous studies have shown that Achn controls developmental decisions in neurons and muscle. In the human mammary gland, Achn expression is restricted to ductal myoepithelial cells. Microarray analysis and immunohistochemistry have shown that Achn expression is elevated in some basal-like ductal carcinomas. To study the possible role of Achn in breast cancer, we engineered human MDA-MB-231 cells to stably express enhanced green fluorescent protein-tagged wild-type Achn (AchnWT), as well as Achn lacking either its nuclear localization signal (AchnNLS) or its nuclear export signal (AchnNES). In in vitro assays, AchnWT and AchnNES, but not AchnNLS, enhanced cell proliferation, lamellipodia formation, and invasive activity and drove expression of the elevated expression of the metastasis-associated proteins MMP-9 and VEGF. To determine if Achn could alter the behavior of human breast cancer cells in vivo, Achn-engineered MDA-MB-231 cells were injected into athymic SCID/Beige mice. AchnWT and AchnNES-expressing tumors displayed enhanced angiogenesis and an approximately 5-fold increase in tumor size relative to either control cells or those expressing AchnNLS. These data suggest that Achn enhances human breast tumor growth and vascularization and that this activity is dependent on nuclear localization.

Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution.

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.

Role of YKL-40 in the angiogenesis, radioresistance, and progression of glioblastoma.

Glioblastoma is one of the most fatal cancers, characterized by a strong vascularized phenotype. YKL-40, a secreted glycoprotein, is overexpressed in patients with glioblastomas and has potential as a novel tumor biomarker. The molecular mechanisms of YKL-40 in glioblastoma development, however, are poorly understood. Here, we aimed to elucidate the role YKL-40 plays in the regulation of VEGF expression, tumor angiogenesis, and radioresistance. YKL-40 up-regulated VEGF expression in glioblastoma cell line U87, and both YKL-40 and VEGF synergistically promote endothelial cell angiogenesis. Interestingly, long term inhibition of VEGF up-regulated YKL-40. YKL-40 induced coordination of membrane receptor syndecan-1 and integrin αvß5, and triggered a signaling cascade through FAK(397) to ERK-1 and ERK-2, leading to elevated VEGF and enhanced angiogenesis. In addition, γ-irradiation of U87 cells increased YKL-40 expression that protects cell death through AKT activation and also enhances endothelial cell angiogenesis. Blockade of YKL-40 activity or expression decreased tumor growth, angiogenesis, and metastasis in xenografted animals. Immunohistochemical analysis of human glioblastomas revealed a correlation between YKL-40, VEGF, and patient survival. These findings have shed light on the mechanisms by which YKL-40 promotes tumor angiogenesis and malignancy, and thus provide a therapeutic target for tumor treatment.