Alyson Evans

Bisphosphonate-induced ATP analog formation and its effect on inhibition of cancer cell growth.

Bisphosphonates (BPs) are effective inhibitors of tumor-induced bone resorption. Recent studies have demonstrated that BPs inhibit growth, attachment and invasion of cancer cells in culture and promote apoptosis. The mechanisms responsible for the observed anti-tumor effects of BPs are beginning to be elucidated. Recently, we reported that nitrogen-containing bisphosphonates (N-BPs) induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of farnesyl diphosphate synthase in the mevalonate pathway. Similar to AppCp-type metabolites of non-N-BPs, ApppI is able to induce apoptosis. This study investigated BP-induced ATP analog formation and its effect on cancer cell growth. To evaluate zoledronic acid (a N-BP)-induced ApppI accumulation, inhibition of protein prenylation and clodronate (a non-N-BP) metabolism to AppCCl2p, MCF-7 and MDA-MB-436 breast cancer cells, MCF-10A nonmalignant breast cells, PC-3 prostate cancer cells, MG-63 osteosarcoma cells, RPMI-8226, and NCI-H929 myeloma cells were treated with 25 μmol/l zoledronic acid or 500 μmol/l clodronate for 24 h. The inhibition of cell growth by zoledronic acid and clodronate was studied in MCF-7, MDA-MB-436, and RPMI-8226 cells by exposing the cells with 1–100 μmol/l zoledronic acid or 10–2000 μmol/l clodronate for 72 h. Marked differences in zoledronic acid-induced ApppI formation and clodronate metabolism between the cancer cell lines were observed. The production of cytotoxic ATP analogs in tumor cells after BP treatment is likely to depend on the activity of enzymes, such as farnesyl diphosphate synthase or aminoacyl-tRNA synthetases, responsible for ATP analog formation. Additionally, the potency of clodronate to inhibit cancer cell growth corresponds to ATP analog formation.

Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo

IntroductionThe majority of deaths from breast cancer are a result of metastases; however, little is understood about the genetic alterations underlying their onset. Genetic profiling has identified the adhesion molecule plakoglobin as being three-fold reduced in expression in primary breast tumors that have metastasized compared with nonmetastatic tumors. In this study, we demonstrate a functional role for plakoglobin in the shedding of tumor cells from the primary site into the circulation.MethodsWe investigated the effects of plakoglobin knockdown on breast cancer cell proliferation, migration, adhesion, and invasion in vitro and on tumor growth and intravasation in vivo. MCF7 and T47D cells were stably transfected with miRNA sequences targeting the plakoglobin gene, or scramble vector. Gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Cell proliferation, adhesion, migration, and invasion were measured by cell counting, flow cytometry, and scratch and Boyden Chamber assays. For in vivo experiments, plakoglobin knockdown and control cells were inoculated into mammary fat pads of mice, and tumor growth, shedding of tumor cells into the bloodstream, and evidence of metastatic bone lesions were monitored with caliper measurement, flow cytometry, and microcomputed tomography (μCT), respectively.ResultsPlakoglobin and γ-catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered after transfection with the scrambled sequence. Reduced plakoglobin resulted in significantly increased in MCF7 and T47D cell proliferation in vitro and in vivo, compared with control, with significantly more tumor cells being shed into the bloodstream of mice bearing plakoglobin knockdown tumors. In addition, plakoglobin knockdown cells showed a >250% increase in invasion through basement membrane and exhibited reduced cell-to-cell adhesion compared with control cells.ConclusionDecreased plakoglobin expression increases the invasive behavior of breast cancer cells. This is the first demonstration of a functional role for plakoglobin/γ-catenin in the metastatic process, indicating that this molecule may represent a target for antimetastatic therapies.

Microvascular Endothelial Cell Responses in vitro and in vivo: Modulation by Zoledronic Acid and Paclitaxel?

Background/Aims: The cytotoxic agent paclitaxel and the anti-resorptive drug zoledronic acid are used in the early and advanced breast cancer setting, respectively. Both agents have been demonstrated to have anti-tumour and anti-endothelial actions. Combining paclitaxel with zoledronic acid induces a synergistic increase in apoptotic breast cancer cell death in vitro, suggesting an increased anti-tumour effect in vivo, but any specific effects on the normal microvasculature and potential side-effects of this combination remain to be established. Methods: The effects of zoledronic acid and paclitaxel were investigated, alone and in combination, on human microvascular endothelial cells in vitro, using functional assays including proliferation, migration, tubule formation and apoptosis. The in vivo effect of the drugs on the normal microvasculature was determined using the dorsal microcirculation chamber model. Results/Conclusion: Zoledronic acid reduced human dermal microvascular endothelial cell (HuDMEC) proliferation, caused accumulation of cells in S phase, and inhibited migration, tube formation and Rap1a prenylation. Paclitaxel significantly inhibited tube formation and proliferation, and increased endothelial necrosis; the combination induced HuDMEC apoptosis and further enhanced the inhibition of tube formation and migration. The combination caused minimal effects on the normal microvasculature in vivo, suggesting that this potential therapeutic strategy is not associated with deleterious microvascular side-effects.

Neoadjuvant Chemotherapy with or without Zoledronic Acid in Early Breast Cancer—A Randomized Biomarker Pilot Study

Purpose: To investigate the short-term biologic effects of neoadjuvant chemotherapy +/− zoledronic acid (ZOL) in invasive breast cancer. Experimental Design: Forty patients were randomized to receive a single 4 mg infusion of ZOL 24 hours after the first cycle of FE100C chemotherapy, or chemotherapy alone. Randomization was stratified for tumor stage, ER, HER2, and menopausal status. All patients had repeat breast core biopsy at day 5 (D5) ± day 21 (D21). Effects on apoptotic index, proliferation (Ki67), growth index, surrogate serum markers of angiogenesis (VEGF), and serum reproductive hormones within the TGFβ family (activin-A, TGFβ1, inhibin-A, and follistatin) were evaluated and compared. Results: Baseline clinicopathologic characteristics were well balanced. Cell growth index (increased apoptosis and reduced proliferation) fell at D5 in both groups but recovered more rapidly with chemotherapy + ZOL compared with chemotherapy alone by D21 (P = 0.006). At D5, a greater reduction in serum VEGF occurred with chemotherapy + ZOL compared with chemotherapy: median percentage change −23.8% [interquartile range (IQR): −32.9 to −15.8] versus −8.4% (IQR: −27.3 to +8.9; P = 0.02), but these effects were lost by D21. Postmenopausal women showed a decrease in follistatin levels from baseline in the chemotherapy + ZOL group at D5 and D21, compared with chemotherapy alone (Pinteraction = 0.051). Conclusions: In this pilot study, short-term changes in biomarkers suggest potentially relevant interactions between tumor biology, chemotherapy, modification of the bone microenvironment, and the endocrine status of the host. Larger studies with more frequent dosing of zoledronic acid are needed to assess these complex interactions more thoroughly. Clin Cancer Res; 19(10); 2755–65. ©2013 AACR.

Erratum to: Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo

Introduction: The majority of deaths from breast cancer are a result of metastases; however, little is understood about the genetic alterations underlying their onset. Genetic profiling has identified the adhesion molecule plakoglobin as being three-fold reduced in expression in primary breast tumors that have metastasized compared with nonmetastatic tumors. In this study, we demonstrate a functional role for plakoglobin in the shedding of tumor cells from the primary site into the circulation. Methods: We investigated the effects of plakoglobin knockdown on breast cancer cell proliferation, migration, adhesion, and invasion in vitro and on tumor growth and intravasation in vivo. MCF7 and T47D cells were stably transfected with miRNA sequences targeting the plakoglobin gene, or scramble vector. Gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Cell proliferation, adhesion, migration, and invasion were measured by cell counting, flow cytometry, and scratch and Boyden Chamber assays. For in vivo experiments, plakoglobin knockdown and control cells were inoculated into mammary fat pads of mice, and tumor growth, shedding of tumor cells into the bloodstream, and evidence of metastatic bone lesions were monitored with caliper measurement, flow cytometry, and microcomputed tomography (μCT), respectively. Results: Plakoglobin and g-catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered after transfection with the scrambled sequence. Reduced plakoglobin resulted in significantly increased in MCF7 and T47D cell proliferation in vitro and in vivo, compared with control, with significantly more tumor cells being shed into the bloodstream of mice bearing plakoglobin knockdown tumors. In addition, plakoglobin knockdown cells showed a >250% increase in invasion through basement membrane and exhibited reduced cell-to-cell adhesion compared with control cells. Conclusion: Decreased plakoglobin expression increases the invasive behavior of breast cancer cells. This is the first demonstration of a functional role for plakoglobin/g-catenin in the metastatic process, indicating that this molecule may represent a target for antimetastatic therapies.