Penelope D. Ottewell

Antitumor Effects of Doxorubicin Followed by Zoledronic Acid in a Mouse Model of Breast Cancer

BACKGROUND The potent antiresorptive drug zoledronic acid (Zol) enhances the antitumor effects of chemotherapy agents in vitro. We investigated the effects of clinically achievable doses of doxorubicin (Dox) and Zol, given alone, in sequence, and in combination, on the growth of established breast tumors in vivo. METHODS Female MF1 nude mice were inoculated subcutaneously with 5 x 10(5) human breast cancer MDA-MB-436 cells that stably expressed green fluorescent protein (ie, MDA-G8 cells). Beginning on day 7 after tumor cell injection, the mice were injected weekly for 6 weeks with saline, Dox (2 mg/kg body weight via intravenous injection), Zol (100 microg/kg body weight via intraperitoneal injection), Dox plus Zol, Zol followed 24 hours later by Dox, or Dox followed 24 hours later by Zol (n = 8-9 mice per group). The effects of treatment on tumor growth were determined by measuring tumor volume; on tumor cell apoptosis and proliferation by immunohistochemistry using antibodies for caspase-3 and Ki-67, respectively; and on bone by microcomputed tomography and bone histomorphometry. All P values are two-sided. RESULTS Treatment with Dox or Zol alone or Zol followed 24 hours later by Dox did not statistically significantly decrease final tumor volume compared with saline. Mice treated with Dox plus Zol had statistically significantly smaller final tumor volumes than those treated with Dox alone (mean = 122 mm(3) vs 328 mm(3), difference = 206 mm(3), 95% confidence interval [CI] = 78 to 335 mm(3), P < .001), with Zol alone (122 mm(3) vs 447 mm(3), difference = 325 mm(3), 95% CI = 197 to 454 mm(3), P < .001), or with Zol followed 24 hours later by Dox (122 mm(3) vs 418 mm(3), difference = 296 mm(3), 95% CI = 168 to 426 mm(3), P < .001). Treatment with Dox followed 24 hours later by Zol almost completely abolished tumor growth. Tumors from mice that were treated with Dox followed by Zol had more caspase-3-positive cells than tumors from mice treated with saline (mean number of caspase-3-positive cells per square millimeter: 605.0 vs 82.19, difference = 522.8, 95% CI = 488.2 to 557.4, P < .001), with Zol alone (605.0 vs 98.44, difference = 506.6, 95% CI = 472.0 to 541.2, P < .001), or with Zol followed by Dox (605.0 vs 103.1, difference = 501.9, 95% CI = 467.3 to 536.5, P < .001). The treatment-induced increase in the number of caspase-3-positive cells was mirrored by a decrease in the number of tumor cells positive for the proliferation marker Ki-67. No evidence of bone disease was detected in any of the treatment groups following microcomputed tomography and histological analysis of bone. CONCLUSION Sequential treatment with Dox followed by Zol elicited substantial antitumor effects in subcutaneous breast tumors in vivo, in the absence of bone disease.

Caspase activation during spontaneous and radiation-induced apoptosis in the murine intestine.

The aim of this study was to characterize the activation of caspase‐3 along the crypt/villus axis in the normal and irradiated intestine and to compare active caspase‐3 expression with existing apoptosis detection techniques. Small and large intestine were removed from mice at various time points after exposure to 8 Gy γ‐radiation. Positive apoptotic cells stained with an antibody against active caspase‐3, haematoxylin and eosin (H&E) or TUNEL were scored in histological sections of small and large intestinal crypts and villi. In the control intestine, active caspase‐3 expression was rarely observed; however, expression was markedly increased following exposure to radiation and was predominantly confined to apoptotic bodies. Measurement of apoptosis in intestinal crypts using active caspase‐3 expression gave similar results to apoptosis detected from H&E‐stained sections. In the normal villus, active caspase‐3 expression was observed infrequently and did not significantly increase following radiation, consistent with a lack of apoptotic body formation from H&E sections. This study indicates that caspase‐3 is activated in intestinal crypts but not in villi following γ‐radiation. Active caspase‐3 detection compared favourably with existing immunological techniques, suggesting that it is a suitable alternative method for apoptosis quantification. Copyright

Differential Effect of Doxorubicin and Zoledronic Acid on Intraosseous versus Extraosseous Breast Tumor Growth In vivo

Purpose: Breast cancer patients with bone metastases are commonly treated with chemotherapeutic agents such as doxorubicin and zoledronic acid to control their bone disease. Sequential administration of doxorubicin followed by zoledronic acid has been shown to increase tumor cell apoptosis in vitro. We have therefore investigated the antitumor effects of clinically relevant doses of these drugs in a mouse model of breast cancer bone metastasis. Experimental Design: MDA-MB-231/BO2 cells were injected via the tail vein into athymic mice. Tumor-induced osteolytic lesions were detected in all animals following X-ray analysis 18 days after tumor cell inoculation (day 18). Mice were administered saline, 100 μg/kg zoledronic acid, 2 mg/kg doxorubicin, doxorubicin and zoledronic acid simultaneously, or doxorubicin followed 24 h later by zoledronic acid. Doxorubicin-treated animals received a second injection on day 25. Tumor growth in the marrow cavity and on the outside surface of the bone was measured as well as tumor cell apoptosis and proliferation. The effects of treatments on bone were evaluated following X-ray and μCT analysis. Results: Sequential treatment with doxorubicin followed by zoledronic acid caused decreased intraosseous tumor burden, which was accompanied by increased levels of tumor cell apoptosis and decreased levels of proliferation, whereas extraosseous parts of the same tumors were unaffected. Administration of zoledronic acid, alone or in combination with doxorubicin, resulted in significantly smaller tumor-induced osteolytic lesions compared with control or doxorubicin-treated animals. Conclusions: This is the first study to show that sequential treatment with clinically relevant doses of doxorubicin, followed 24 h later by zoledronic acid, reduces intraosseous but not extraosseous growth of BO2 breast tumors. Our results suggest that breast cancer patients with metastatic bone disease may benefit from sequential treatment using doxorubicin and zoledronic acid.

Zoledronic Acid Has Differential Antitumor Activity in the Pre- and Postmenopausal Bone Microenvironment In Vivo

Purpose: Clinical trials in early breast cancer have suggested that benefits of adjuvant bone-targeted treatments are restricted to women with established menopause. We developed models that mimic pre- and postmenopausal status to investigate effects of altered bone turnover on growth of disseminated breast tumor cells. Here, we report a differential antitumor effect of zoledronic acid (ZOL) in these two settings. Experimental design: Twleve-week-old female Balb/c-nude mice with disseminated MDA-MB-231 breast tumor cells in bone underwent sham operation or ovariectomy (OVX), mimicking the pre- and postmenopausal bone microenvironment, respectively. To determine the effects of bone-targeted therapy, sham/OVX animals received saline or 100 μg/kg ZOL weekly. Tumor growth was assessed by in vivo imaging and effects on bone by real-time PCR, micro-CT, histomorphometry, and measurements of bone markers. Disseminated tumor cells were detected by two-photon microscopy. Results: OVX increased bone resorption and induced growth of disseminated tumor cells in bone. Tumors were detected in 83% of animals following OVX (postmenopausal model) compared with 17% following sham operation (premenopausal model). OVX had no effect on tumors outside of bone. OVX-induced tumor growth was completely prevented by ZOL, despite the presence of disseminated tumor cells. ZOL did not affect tumor growth in bone in the sham-operated animals. ZOL increased bone volume in both groups. Conclusions: This is the first demonstration that tumor growth is driven by osteoclast-mediated mechanisms in models that mimic post- but not premenopausal bone, providing a biologic rationale for the differential antitumor effects of ZOL reported in these settings. Clin Cancer Res; 20(11); 2922–32. ©2014 AACR.

From genetic abnormality to metastases: murine models of breast cancer and their use in the development of anticancer therapies

SummaryNumerous mouse models of mammary cancer have been developed that mimic selective aspects of human disease. The use of these models has enabled preclinical chemotherapeutic, chemoprevention, and genetic therapy studies in vivo, the testing of gene delivery systems, and the identification of tumour and metastasis suppressor and inducer genes. This review has discussed the most abundantly used murine models of mammary cancer including: spontaneous tumours, chemically induced tumours, orthotopic and syngeneic tumour transplantation, injected tumours, and genetically engineered mice with a predisposition to neoplasia. Each model has been discussed with regards to its merits and limitations for investigating the genetic and phenotypic alterations involved in the human disease as well as its potential usefulness for the development of new treatment strategies. To date no single mouse model is available with the ability to replicate the entire disease process, however, existing models continue to provide invaluable insights into breast cancer induction and progression that would be impossible to obtain using in vitro models alone.

Anticancer mechanisms of doxorubicin and zoledronic acid in breast cancer tumor growth in bone

Patients with advanced breast cancer frequently develop bone metastases, and at this stage, the disease is considered incurable. Here, we show that a 6-week course of weekly administration of doxorubicin (2 mg/kg), followed 24 hours later by the bisphosphonate zoledronic acid (100μg/kg), causes substantial inhibition of MDA-MB-436 breast tumor burden in bone of immunocompromised mice, compared with administration of the single agents. Molecular analysis of tumors from animals treated sequentially with doxorubicin followed by zoledronic acid showed reduced numbers of proliferating tumor cells and decreased expression of cyclins E1, B, D1, and D3 as well as cdk2 and cdk4. Tumors from the sequential treatment group also displayed increased levels of apoptosis, increased expression of bcl2-associated X protein, decreased expression of B-cell chronic lymphocytic leukemia/lymphoma 2, and activation of caspase 3, 8, and 9. Zoledronic acid caused a small reduction in tumor volume, reduced tumor cell proliferation, and decreased expression of cyclins D1 and D3, compared with tumors from animals treated with saline or doxorubicin. Doxorubicin had no effect on tumor growth, cell cycle, or apoptosis in vivo, but did cause increased accumulation of a bisphosphonate in MDA-MB-436 cells in vitro, suggesting that doxorubicin may affect subsequent uptake of zoledronic acid. In support of this, accumulation of unprenylated Rap1A, a surrogate marker of zoledronic acid, was only detected in tumors following sequential treatment, and not following treatment with zoledronic acid alone. Our data are the first to show the specific molecular pathways by which sequential treatment with doxorubicin and zoledronic acid induce tumor cell apoptosis and inhibit proliferation in an in vivo model of breast tumor growth in bone. [Mol Cancer Ther 2009;8(10):2821–32]

Prostate Cancer Cells Preferentially Home to Osteoblast-rich Areas in the Early Stages of Bone Metastasis: Evidence From In Vivo Models

It has been suggested that metastasis‐initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or “niche”). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non‐uniform, and we hypothesize that studying co‐localization of individual tumor cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3‐NW1, LN‐CaP, and C4 2B4) colonizing the tibiae of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labeled with the fluorescent cell membrane dye (Vybrant DiD) were found by two‐photon microscopy to be engrafted in the tibiae in close proximity (∼40 µm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5‐fold higher number of osteoblasts and 7‐fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C‐X‐C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast‐rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases.

Castration-induced bone loss triggers growth of disseminated prostate cancer cells in bone

Up to 90% of patients with castrate-resistant prostate cancer develop bone metastases, and the majority of these men have received androgen deprivation therapy known to cause bone loss. Whether this treatment-induced change to the bone microenvironment affects disseminated tumour cells, potentially stimulating development of bone metastasis, remains to be determined. The objective of this study was to use an in vivo model mimicking androgen ablation to establish the effects of this intervention on disseminated prostate cancer cells in bone. We mimicked the effects of androgen deprivation on bone metastasis by castrating 12-week-old BALB/c nude mice that had disseminated, hormone-insensitive PC3 prostate cancer cells present in the long bones. Castration caused increased bone resorption and loss of bone volume, compared with sham operation. In addition, castration triggered growth of disseminated PC3 cells to form bone metastasis in 70% of animals. In contrast, only 10% of sham-operated animals had detectable long bone tumours. Weekly administration of 100 μg/kg zoledronic acid (ZOL) prevented castration-induced tumour growth in bone and increased bone volume, but did not eliminate the disseminated tumour cells. ZOL had no effect on tumour growth in the sham-operated animals, despite causing a significant increase in bone volume. This is the first demonstration that, in a model of prostate cancer bone metastasis, mimicking androgen ablation results in growth of disseminated tumour cells in bone through osteoclast-mediated mechanisms. We provide the first biological evidence supporting the administration of ZOL to prostate cancer patients at the time of androgen ablation to prevent subsequent relapse in bone.

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.

Progastrin stimulates murine colonic epithelial mitosis after DNA damage

Abstract Background & Aims: Transgenic mice that overexpress progastrin are more susceptible than either wild-type mice or mice that overexpress amidated gastrin to chemical carcinogen-induced colonic adenomas. We have investigated whether alterations in the regulation of apoptosis or mitosis after DNA damage contribute to the effects of progastrin on murine colonic epithelium. Methods: Apoptosis and mitosis were assessed on a cell positional basis in murine intestinal epithelium after γ-irradiation. Mice analyzed were progastrin overexpressing, gastrin overexpressing, gastrin knockout, and their wild-type counterparts. The expression of cell cycle regulators was analyzed by gene array and Western blotting. Results: Apoptosis was induced to similar levels in the small intestinal and colonic crypts of all mice 4.5 hours after 8 Gy γ-radiation. Colonic mitosis was inhibited to almost undetectable levels by 8Gy γ-radiation in wild-type, gastrin-knockout, and gastrin-overexpressing mice. However, significant colonic mitosis persisted in progastrin-overexpressing mice up to 24 hours after 8Gy γ-radiation. Increased levels of cdk4 and cyclin D1 proteins were found in the colonic epithelium of progastrin-overexpressing mice relative to wild-type animals after γ-radiation. Conclusions: After DNA damage by γ-radiation, mice with elevated progastrin exhibit significantly higher levels of colonic mitosis than wild-type or gastrin-overexpressing mice. Persistently elevated cdk4 and cyclin D1 in progastrin overexpressing mice accounts for the capacity of colon cells to continue with the cell cycle after DNA damage.