Vascular endothelial growth factor (VEGF) plays key roles in angiogenesis, vasculogenesis, and wound healing. In cancers, including triple negative breast cancer (TNBC), VEGF has been associated with ...increased invasion and metastasis, processes that require cancer cells to traverse through the extracellular matrix (ECM) and establish angiogenesis at distant sites. To further understand the role of VEGF in modifying the ECM, we characterized VEGF-mediated changes in the ECM of tumors derived from TNBC MDA-MB-231 cells engineered to overexpress VEGF. We established that increased VEGF expression by these cells resulted in tumors with reduced collagen 1 (Col1) fibers, fibronectin, and hyaluronan. Molecular characterization of tumors identified an increase of MMP1, uPAR, and LOX, and a decrease of MMP2, and ADAMTS1. α-SMA, a marker of cancer associated fibroblasts (CAFs), increased, and FAP-α, a marker of a subset of CAFs associated with immune suppression, decreased with VEGF overexpression. Analysis of human data from The Cancer Genome Atlas Program confirmed mRNA differences for several molecules when comparing TNBC with high and low VEGF expression. We additionally characterized enzymatic changes induced by VEGF overexpression in three different cancer cell lines that clearly identified autocrine-mediated changes, specifically uPAR, in these enzymes. Unlike the increase of Col1 fibers and fibronectin mediated by VEGF during wound healing, in the TNBC model, VEGF significantly reduced key protein components of the ECM. These results further expand our understanding of the role of VEGF in cancer progression and identify potential ECM-related targets to disrupt this progression.
Metastatic dissemination continues to be a major cause of prostate cancer (PCa) mortality, creating a compelling need to understand factors that play a role in the metastatic cascade. Since hypoxia ...plays an important role in PCa aggressiveness, we characterized patterns of hypoxia in the primary tumor and metastatic environments of a human PCa xenograft. We previously developed and characterized an imaging strategy based on the hypoxia response element (HRE)–driven expression of long-lived enhanced green fluorescent protein (EGFP) and short-lived luciferase (luc) fused to the oxygen-dependent degradation domain in human PCa PC-3 cells. Both reporter proteins were placed under the transcriptional control of a five–tandem repeat HRE sequence. PC-3 cells also constitutively expressed the tdTomato red fluorescent protein, allowing cancer cell detection in vivo. This “timer” strategy can provide information on the temporal evolution of HIF activity and hypoxia in tumors. Here, for the first time, we performed in vivo and ex vivo imaging of this dual HIF reporter system in PC-3 metastatic tumors implanted orthotopically in the prostate and PC-3 nonmetastatic tumors implanted subcutaneously. We observed distinct patterns of EGFP and luc expression in subcutaneous and orthotopic tumors, and in metastatic nodules, that provide new insights into the presence of hypoxia at primary and metastatic tumor sites, and of the role of hypoxia in metastasis.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Developed a hypoxia theranostic imaging strategy to eliminate hypoxic cells.•Hypoxic cell elimination resulted in fewer cancer associated fibroblasts (CAFs)•Collagen 1 fiber patterns were altered ...with hypoxic cell elimination.•cDNA nanoparticles with HRE driven prodrug enzyme expression can target hypoxia.
Hypoxia is frequently observed in human prostate cancer, and is associated with chemoresistance, radioresistance, metastasis, and castrate-resistance. Our purpose in these studies was to perform hypoxia theranostics by combining in vivo hypoxia imaging and hypoxic cancer cell targeting in a human prostate cancer xenograft. This was achieved by engineering PC3 human prostate cancer cells to express luciferase as well as a prodrug enzyme, yeast cytosine deaminase, under control of hypoxic response elements (HREs). Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to HRE in the promoter region of target gene that results in their increased transcription. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation. HRE-driven luciferase expression allowed us to detect hypoxia in vivo to time the administration of the nontoxic prodrug 5-fluorocytosine that was converted by yeast cytosine deaminase, expressed under HRE regulation, to the chemotherapy agent 5-fluorouracil to target hypoxic cells. Conversion of 5-fluorocytosine to 5-fluorouracil was detected in vivo by 19F magnetic resonance spectroscopy. Morphological and immunohistochemical staining and molecular analyses were performed to characterize tumor microenvironment changes in cancer-associated fibroblasts, cell viability, collagen 1 fiber patterns, and HIF-1α. These studies expand our understanding of the effects of eliminating hypoxic cancer cells on the tumor microenvironment and in reducing stromal cell populations such as cancer-associated fibroblasts.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
An increase of cellular phosphocholine (PC) and total choline (tCho)-containing compounds as well as alterations in lipids have been consistently observed in cancer cells and tissue. These metabolic ...changes are closely related to malignant transformation, invasion, and metastasis. The study of cancer cells in culture plays an important role in understanding mechanisms leading to altered choline (Cho) and lipid metabolism in cancer, as it provides a carefully controlled environment. However, a solid tumor is a complex system with a unique tumor microenvironment frequently containing hypoxic and acidic regions and areas of nutrient deprivation and necrosis. Cancer cell-stromal cell interactions and the extracellular matrix may also alter Cho and lipid metabolism. Human tumor xenograft models in mice are useful to mimic the growth of human cancers and provide insights into the influence of
conditions on metabolism. Here, we have compared metabolites, obtained with high resolution
H MRS of extracts from human breast and prostate cancer cells in a 2-dimensional (2D) monolayer culture and from solid tumor xenografts derived from these cells, as well as the protein expression of enzymes that regulate Cho and lipid metabolism. Our data demonstrate significant differences in Cho and lipid metabolism and protein expression patterns between human breast and prostate cancer cells in culture and in tumors derived from these cells. These data highlight the influence of the tumor microenvironment on Cho and lipid metabolism.
Hypoxia inducible factors (HIFs) are transcription factors that mediate the response of cells to hypoxia. HIFs have wide-ranging effects on metabolism, the tumor microenvironment (TME) and the ...extracellular matrix (ECM). Here we investigated the silencing effects of two of the three known isoforms, HIF-1α and HIF-2α, on collagen 1 (Col1) fibers, which form a major component of the ECM of tumors. Using a loss-of-function approach for HIF-1α or 2α or both HIF-1α and 2α, we identified a relationship between HIFs and Col1 fibers in MDA-MB-231 tumors. Tumors derived from MDA-MB-231 cells with HIF-1α or 2α or both HIF-1α and 2α silenced contained higher percent fiber volume and lower inter-fiber distance compared to tumors derived from empty vector MDA-MB-231 cells. Depending upon the type of silencing, we observed changes in Col1 degrading enzymes, and enzymes involved in Col1 synthesis and deposition. Additionally, a reduction in lysyl oxidase protein expression in HIF-down-regulated tumors suggests that more non-cross-linked fibers were present. Collectively these results identify the role of HIFs in modifying the ECM and the TME and provide new insights into the effects of hypoxia on the tumor ECM.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying ...molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The ability to destroy cancer cells while sparing normal tissue is highly sought after in cancer therapy. Small interfering RNA (siRNA)-mediated silencing of cancer-cell-specific targets and the use ...of a prodrug enzyme delivered to the tumor to convert a nontoxic prodrug to an active drug are two promising approaches in achieving this goal. Combining both approaches into a single treatment strategy can amplify selective targeting of cancer cells while sparing normal tissue. Noninvasive imaging can assist in optimizing such a strategy by determining effective tumor delivery of the siRNA and prodrug enzyme to time prodrug administration and detecting target down-regulation by siRNA and prodrug conversion by the enzyme. In proof-of-principle studies, we synthesized a nanoplex carrying magnetic resonance imaging (MRI) reporters for in vivo detection and optical reporters for microscopy to image the delivery of siRNA and a functional prodrug enzyme in breast tumors and achieve image-guided molecular targeted cancer therapy. siRNA targeting of choline kinase-α (Chk-α), an enzyme significantly up-regulated in aggressive breast cancer cells, was combined with the prodrug enzyme bacterial cytosine deaminase (bCD) that converts the nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU). In vivo MRI and optical imaging showed efficient intratumoral nanoplex delivery. siRNA-mediated down-regulation of Chk-α and the conversion of 5-FC to 5-FU by bCD were detected noninvasively with 1H MR spectroscopic imaging and 19F MR spectroscopy. Combined siRNA and prodrug enzyme activated treatment achieved higher growth delay than either treatment alone. The strategy can be expanded to target multiple pathways with siRNA.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Introduction
Malignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating ...symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis.
Objective
Our purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer.
Method
We determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8- vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution
1
H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with
1
H NMR.
Results
Distinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium.
Conclusion
High-resolution
1
H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.
Elevated phosphocholine (PC) and total choline (tCho) metabolites are widely established characteristics of most cancer cells, including breast cancer. Effective silencing of choline kinase (chk), ...the enzyme that converts choline to PC, is associated with reduced tumor growth. The functional importance and down-regulation of chk using RNA interference has been previously established. Here, we report on the preclinical evaluation of lentiviral vector-mediated down-regulation of chk using short hairpin RNA (shRNA) in established tumors derived from human breast cancer cells. Concentrated lentivirus expressing shRNA against chk was injected i.v. in the tail vein of MDA-MB-231 tumor-bearing female severe combined immunodeficient mice. Transduction efficiency in cells and tumors in vivo was assessed optically by enhanced green fluorescent protein expression and additionally from chk mRNA and protein levels. An 80% reduction in chk mRNA and protein was achieved following approximately 90% transduction efficiency in cells. After transduction with chk-shRNA, (1)H magnetic resonance spectroscopy (MRS) of cell and tumor extracts showed decreases in PC and tCho levels (P < 0.01 and 0.05, respectively) in comparison with controls. PC levels were monitored noninvasively by (31)P MRS in tumors and by (1)H MRS in cell and tumor tissue extracts. Noninvasive (31)P MR spectra of chk-shRNA-transduced tumors in vivo showed lower PC and phosphomonoester levels that were associated with reduced tumor growth and proliferation. This study shows the use of lentiviral vectors to target chk in a human breast cancer xenograft and noninvasive MRS detection of this targeting.
PDF
