Yelena Mironchik

Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

Oncogenic role of DDX3 in breast cancer biogenesis

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.

Metabolic Imaging of Pancreatic Ductal Adenocarcinoma Detects Altered Choline Metabolism

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets. Experimental Design: We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets. Results: We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between immortalized human pancreatic cells and neoplastic PDAC cells. Molecular characterization revealed overexpression of choline kinase (Chk)-α, choline transporter 1 (CHT1), and choline transporter–like protein 1 (CTL1) in the PDAC cell lines and tumors. Conclusions: Collectively, these data identify new metabolic characteristics of PDAC and reveal potential metabolic targets. Total choline detected with 1H MRSI may provide an intrinsic, imaging probe–independent biomarker to complement existing techniques in detecting PDAC. The expression of Chk-α, CHT1, and CTL1 may provide additional molecular markers in aspirated cytological samples. Clin Cancer Res; 21(2); 386–95. ©2014 AACR.

Phototheranostics of CD44-positive cell populations in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer that has limited treatment options. Its high rates of recurrence and metastasis have been associated, in part, with a subpopulation of breast cancer stem-like cells that are resistant to conventional therapies. A compendium of markers such as CD44high/CD24low, and increased expression of the ABCG2 transporter and increased aldehyde dehydrogenase (ALDH1), have been associated with these cells. We developed a CD44-targeted monoclonal antibody photosensitizer conjugate for combined fluorescent detection and photoimmunotherapy (PIT) of CD44 expressing cells in TNBC. The CD44-targeted conjugate demonstrated acute cell killing of breast cancer cells with high CD44 expression. This cell death process was dependent upon CD44-specific cell membrane binding combined with near-infrared irradiation. The conjugate selectively accumulated in CD44-positive tumors and caused dramatic tumor shrinkage and efficient elimination of CD44-positive cell populations following irradiation. This novel phototheranostic strategy provides a promising opportunity for the destruction of CD44-positive populations that include cancer stem-like cells, in locally advanced primary and metastatic TNBC.

HIF isoforms have divergent effects on invasion, metastasis, metabolism and formation of lipid droplets

Cancer cells adapt to hypoxia by the stabilization of hypoxia inducible factor (HIF)-α isoforms that increase the transcription of several genes. Among the genes regulated by HIF are enzymes that play a role in invasion, metastasis and metabolism. We engineered triple (estrogen receptor/progesterone receptor/HER2/neu) negative, invasive MDA-MB-231 and SUM149 human breast cancer cells to silence the expression of HIF-1α, HIF-2α or both isoforms of HIF-α. We determined the metabolic consequences of HIF silencing and the ability of HIF-α silenced cells to invade and degrade the extracellular matrix (ECM) under carefully controlled normoxic and hypoxic conditions. We found that silencing HIF-1α alone was not sufficient to attenuate invasiveness in both MDA-MB-231 and SUM149 cell lines. Significantly reduced metastatic burden was observed in single (HIF-1α or HIF-2α) and double α-isoform silenced cells, with the reduction most evident when both HIF-1α and HIF-2α were silenced in MDA-MB-231 cells. HIF-2α played a major role in altering cell metabolism. Lipids and lipid droplets were significantly reduced in HIF-2α and double silenced MDA-MB-231 and SUM149 cells, implicating HIF in their regulation. In addition, lactate production and glucose consumption were reduced. These results suggest that in vivo, cells in or near hypoxic regions are likely to be more invasive. The data indicate that targeting HIF-1α alone is not sufficient to attenuate invasiveness, and that both HIF-1α and HIF-2α play a role in the metastatic cascade in these two cell lines.

Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts

Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers. COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel timer imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.

Structure and function of a prostate cancer dissemination-permissive extracellular matrix

Purpose: The poor prognosis of metastatic prostate cancer continues to present a major challenge in prostate cancer treatment. The tumor extracellular matrix (ECM) plays an important role in facilitating metastasis. Here, we investigated the structure and function of an ECM that facilitates prostate cancer metastasis by comparing orthotopic tumors that frequently metastasize to poorly metastatic subcutaneous tumors. Experimental Design: Both tumors were derived from a human prostate cancer PC3 cell line engineered to fluoresce under hypoxia. Second harmonic generation (SHG) microscopy was used to characterize collagen 1 (Col1) fiber patterns in the xenografts as well as in human samples. MRI was used to determine albumin-Gd-diethylenetriaminepenta-acetate (alb-GdDTPA) transport through the ECM using a saturation recovery MR method combined with fast T1 SNAPSHOT-FLASH imaging. Cancer-associated fibroblasts (CAF) were also quantified in these tumors. Results: Significant structural and functional differences were identified in the prometastatic orthotopic tumor ECM compared to the less metastatic subcutaneous tumor ECM. The significantly higher number of CAFs in orthotopic tumors may explain the higher Col1 fiber volumes in these tumors. In vivo, alb-GdDTPA pooling was significantly elevated in metastatic orthotopic tumors, consistent with the increased Col1 fibers. Conclusions: Developing noninvasive MRI indices of macromolecular transport, together with characterization of Col1 fiber patterns and CAFs can assist in stratifying prostate cancers for aggressive treatments or active surveillance. These results highlight the role of CAFs in supporting or creating aggressive cancers, and the importance of depleting CAFs to prevent metastatic dissemination in prostate cancer. Clin Cancer Res; 23(9); 2245–54. ©2016 AACR.

Abstract 5348: Transduction of tumors in vivo with choline kinase shRNA downregulates breast cancer stem cell markers

Increased phosphocholine is one of the metabolic signatures of cancer, and is closely associated with malignant transformation, invasion and metastasis. Increased phosphocholine is primarily due to increased expression of choline kinaseα, the enzyme that converts choline to the membrane precursor phosphocholine. Stem-like breast cancer cells (SBCCs) are drug resistant, invasive, and likely to lead to recurrence and repopulation. High CD44 adhesion molecule expression and high expression of the drug transporter ABCG2 are two markers associated with populations enriched with SBCCs [1]. Here we have examined the relationship between choline kinase and these two markers of SBCCs. We have determined the relationship between choline kinase downregulation and two markers of SBCCs, CD44 and ABCG2 in MDA-MB-231 breast cells in vitro and tumors in vivo upon lentiviral transduction of shRNA targeting choline kinase [2]. CD44 and its variant isoforms CD44v6 and CD44v8, together with ABCG2 showed a significant reduction in mRNA (30% for CD44 total; 80% for CDv6 and v8, and 45% for ABCG2) and protein expression levels following transduction of MDA-MB-231 cells with lentivirus expressing shRNA against choline kinase. Similar decreases were also observed following systemic intravenous injections of lentivirus expressing choline kinase shRNA delivered over four weeks. These studies provide insight into potential metabolic targeting of SBCCs. 1. Al-Hajj, M et al., Proc Natl Acad Sci U S A, 2003. 2. Krishnamachary, B et al., Cancer Research, 2009. This work was supported by NIH R01CA136576 and P50 CA103175. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5348. doi:1538-7445.AM2012-5348

Metabolic consequences of HIF silencing in a triple negative human breast cancer xenograft

Hypoxia is frequently encountered in tumors and results in the stabilization of hypoxia inducible factors (HIFs). These factors transcriptionally activate genes that allow cells to adapt to hypoxia. In cancers, hypoxia and HIFs have been associated with increased invasion, metastasis, and resistance to chemo and radiation therapy. Here we have characterized the metabolic consequences of silencing HIF-1α and HIF-2α singly or combined in MDA-MB-231 triple negative human breast cancer xenografts, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI) of in vivo tumors, and high-resolution 1H MRS of tumor extracts. Tumors from all three sublines showed a significant reduction of growth rate. We identified new metabolic targets of HIF, and demonstrated the divergent consequences of silencing HIF-1α and HIF-2α individually on some of these targets. These data expand our understanding of the metabolic pathways regulated by HIFs that may provide new insights into the adaptive metabolic response of cancer cells to hypoxia. Such insights may lead to novel metabolism based therapeutic targets for triple negative breast cancer.