Lori Rice

Impact of the SRC inhibitor dasatinib on the metastatic phenotype of human prostate cancer cells

SRC, a non-receptor tyrosine kinase, is frequently over-expressed and highly activated in blood as well as solid tumors in various organs, including prostate, and has been associated with aggressive disease and a poor patient prognosis. Prostate cancer patients with a high risk of developing metastases have few treatment options, none of which can result in a durable cure. Therefore, the aim of the present study was to examine the impact of a SRC inhibitor, dasatinib, on the ability of human prostate cancer cell to complete key steps in the metastatic process, including invasion and angiogenesis. Dasatinib treatment impaired the metastatic phenotypes of the human prostate cancer cell lines, PC-3, DU-145, and LNCaP, by significantly reducing migration and invasion in modified Boyden chambers. Inhibition of phosphorylation, and therefore enhanced activation, of SRC and key downstream signaling pathway elements, including FAK, STAT3, Paxillin, and Akt, as determined by Western blotting, also was observed. This suggests that dasatinib interferes with critical cell functions associated with the metastatic cascade. Dasatinib also had direct effects on the ability of microvascular endothelial cells to form tubes in vitro and impaired the ability of PC-3 cells to induce angiogenesis in vivo. In conclusion, the present findings suggest that SRC inhibition by dasatinib may have utility in reducing the metastatic spread of prostate cancer cells.

Support of a Free Radical Mechanism for Enhanced Antitumor Efficacy of the Microtubule Disruptor OXi4503

Unlike normal blood vessels, the unique characteristics of an expanding, disorganized and leaky tumor vascular network can be targeted for therapeutic gain by vascular disrupting agents (VDAs), which promote rapid and selective collapse of tumor vessels, causing extensive secondary cancer cell death. A hallmark observation following VDA treatment is the survival of neoplastic cells at the tumor periphery. However, comparative studies with the second generation tubulin-binding VDA OXi4503 indicate that the viable rim of tumor tissue remaining following treatment with this agent is significantly smaller than that seen for the lead VDA, combretastatin. OXi4503 is the cis-isomer of CA1P and it has been speculated that this agents increased antitumor efficacy may be due to its reported metabolism to orthoquinone intermediates leading to the formation of cytotoxic free radicals. To examine this possibility in situ, KHT sarcoma-bearing mice were treated with either the cis- or trans-isomer of CA1P. Since both isomers can form quinone intermediates but only the cis-isomer binds tubulin, such a comparison allows the effects of vascular collapse to be evaluated independently from those caused by the reactive hydroxyl groups. The results showed that the cis-isomer (OXi4503) significantly impaired tumor blood flow leading to secondary tumor cell death and >95% tumor necrosis 24h post drug exposure. Treatment with the trans-isomer had no effect on these parameters. However, the combination of the trans-isomer with combretastatin increased the antitumor efficacy of the latter agent to near that of OXi4503. These findings indicate that while the predominant in vivo effect of OXi4503 is clearly due to microtubule collapse and vascular shut-down, the formation of toxic free radicals likely contributes to its enhanced potency.

Transcriptional repression of VEGF by ZNF24: mechanistic studies and vascular consequences in vivo

VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative regulator of tumor growth by inhibiting angiogenesis.

Cathepsin L inactivation leads to multimodal inhibition of prostate cancer cell dissemination in a preclinical bone metastasis model

It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra‐cardiac injection of human PC‐3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients.

MRI-Based Computational Model of Heterogeneous Tracer Transport following Local Infusion into a Mouse Hind Limb Tumor

Systemic drug delivery to solid tumors involving macromolecular therapeutic agents is challenging for many reasons. Amongst them is their chaotic microvasculature which often leads to inadequate and uneven uptake of the drug. Localized drug delivery can circumvent such obstacles and convection-enhanced delivery (CED) - controlled infusion of the drug directly into the tissue - has emerged as a promising delivery method for distributing macromolecules over larger tissue volumes. In this study, a three-dimensional MR image-based computational porous media transport model accounting for realistic anatomical geometry and tumor leakiness was developed for predicting the interstitial flow field and distribution of albumin tracer following CED into the hind-limb tumor (KHT sarcoma) in a mouse. Sensitivity of the model to changes in infusion flow rate, catheter placement and tissue hydraulic conductivity were investigated. The model predictions suggest that 1) tracer distribution is asymmetric due to heterogeneous porosity; 2) tracer distribution volume varies linearly with infusion volume within the whole leg, and exponentially within the tumor reaching a maximum steady-state value; 3) infusion at the center of the tumor with high flow rates leads to maximum tracer coverage in the tumor with minimal leakage outside; and 4) increasing the tissue hydraulic conductivity lowers the tumor interstitial fluid pressure and decreases the tracer distribution volume within the whole leg and tumor. The model thus predicts that the interstitial fluid flow and drug transport is sensitive to porosity and changes in extracellular space. This image-based model thus serves as a potential tool for exploring the effects of transport heterogeneity in tumors.

Abstract 5211: In vivo fluorescence and spectral microscopy of the effects of aerobic exercise on tumor oxygenation and perfusion in breast cancer

Abnormal tumor angiogenesis leads to inefficient blood flow and inhibited delivery of oxygen, resulting in a hypoxic tumor microenvironment. In radiation therapy the presence of oxygen is necessary to impart lasting damage to cancer cells, therefore the modulation of tumor hypoxia has been an important focus to improve radiation therapy outcome. While strategies such as hyperbaric chambers and hypoxic cell radiosensitizers or cytotoxins have shown merit, their success in the clinic has been limited. A potential method to increase oxygenation and decrease hypoxia in a tumor mass that is safe and well-tolerated is aerobic exercise. For cancer patients, the benefits of exercise in a palliative setting are well known, however the effects of exercise on tumor microvasculature and blood flow have not been extensively characterized. In vivo microscopy of microvessel function in preclinical models is a useful tool for the study of tumor vascular structure and function. The combination of hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence (FPF) imaging of blood transit time in the murine dorsal skinfold window chamber has been previously used to characterize tumor angiogenesis, wound healing and the response of tumor microvasculature to vascular targeting agents. Hb saturation imaging reveals the oxygenation of blood within microvessels while FPF imaging gives information regarding blood flow and network connections by recording a fluorescent contrast agent as it is injected into circulation. In the present study, this combination imaging technique is used to evaluate the impact of treadmill running on tumor blood flow and microvessel oxygenation in real time. 4T1 mammary tumors were initiated in nude mice bearing titanium window chambers. Once tumors reached a volume of ∼5mm3, mice were subjected to daily treadmill running for 5-7 consecutive days (30 min, 15-18 m/min, 10° incline). As a sedentary control, mice were placed on a stationary treadmill for equivalent periods of time. Combination imaging was performed daily to monitor microvessel oxygenation and blood flow. On the first day of exercise, imaging was performed both before and after animals were exercised. Preliminary results indicate that following one bout of exercise, tumor perfusion is increased, however there was no significant correlation between exercise and tumor microvessel oxygenation. Citation Format: Jennifer A. Lee, Jennifer M. Wiggins, Lori P. Rice, Dietmar W. Siemann. In vivo fluorescence and spectral microscopy of the effects of aerobic exercise on tumor oxygenation and perfusion in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2015-5211

Abstract 5913: The impact of daily exercise on tumor perfusion

Aberrant blood vessel networks in solid tumors lead to impaired tissue perfusion and areas of hypoxia (pO2 Citation Format: Jennifer M. Wiggins, Sharon Lepler, Christine Pampo, Lori Rice, Jennifer A. Lee, Dietmar Siemann. The impact of daily exercise on tumor perfusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5913. doi:10.1158/1538-7445.AM2017-5913

Abstract 732: The impact of aerobic exercise on the tumor microenvironment

Intratumoral hypoxia (pO2 The goal of the present investigations was to determine whether aerobic exercise could be applied to improve tumor perfusion, oxygenation and the immune anti-tumor response in breast cancer and fibrosarcoma models. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy. The effects of mild and moderate intensity treadmill running were studied in mice bearing syngeneic murine mammary carcinomas (4T1 and EMT6) or fibrosarcomas (KHT). The exercise intensities were determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and exercise commenced when tumors reached a size of ∼500 mm3 (single) or ∼200 mm3 (repeated bouts). Once size was attained, mice were exposed to either mild (12 m/min) or moderate (18 m/min) intensity exercise. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of the exercise period tumors were analyzed by histology to assess for physiological changes. Specifically, blood was collected and tumors were harvested, sectioned and evaluated by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion and while the hypoxia marker (EF5) was used to determine the level of tumor hypoxia. Both markers were quantified using a Chalkley counter and ImageJ NIH software. In addition, the evaluation of tumor infiltrating immune cells after exercise compared to rest is under active investigation by histological analysis. Furthermore, plasma samples from exercised mice are being tested for immune related cytokines, chemokines and growth factors and compared to those from sedentary controls. Results to date indicate no difference in tumor growth rate between sedentary and exercising mice. Mild daily bouts of aerobic exercise do not affect the total number of tumor blood vessels but do increase the number of blood vessels that are actively perfused. These findings suggest that exercise may have potential utility in overcoming the aberrant microenvironmental conditions associated in solid tumors with therapeutic resistance. Citation Format: Jennifer M. Wiggins, Jennifer Lee, Lori Rice, Sharon Lepler, Christine Pampo, Dietmar Siemann. The impact of aerobic exercise on the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 732.

Abstract 777: Effects of Src inhibitors and soy isoflavones on human prostate cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Src family kinases are often over-expressed and highly active in solid tumors, including prostate cancer. This phenotype is associated with a poor prognosis partly because Src is a key factor in important signaling pathways involved in cell proliferation, angiogenesis, and initiation of metastasis. As a result, several promising small molecule targeting agents have been developed to prevent phosphorylation of key tyrosine residues that produce Src activation. However, these agents are not without side effects. The purpose of this study was to determine if adding a cytostatic soy isoflavone extract (ISF) to the treatment would produce optimal results at lower doses of the Src inhibitor. The ISF used in these studies has been shown to inhibit tumor growth in mice carrying human prostate cancer cell xenografts without causing toxicity to the host. Methods: Aggressive PC-3ML cells (a gift from A. Fatatis, Drexel University), and weakly tumorigenic LNCaP cells were treated for 24 hr with various concentrations of Src inhibitors (dasatinib, saracatinib), ISF (200 ug/ml, NovaSoy), or a combination. To evaluate effects on the metastatic potential of the cells, functional assays of cell growth and motility were performed, including those that assess clonogenic cell survival, cell cycle progression, and transwell migration and invasion activity. Results: Src inhibitors and ISF alone produced very little reduction in cell viability, but significant cytostatic effects, as determined by a reduction in clonogenicity, the number of cells able to form 50-cell colonies. This was likely due, in part, to changes in cell cycle progression. Both dasatinib and saracatinib caused an accumulation of cells in the G1 phase. As expected, ISF treatment resulted in higher numbers of cells in the G2/M phase. When the treatments were combined, cells exposed to dasatinib were observed to accumulate in both phases, with a significant decrease in S-phase cells. The results were not significant for saracatinib-treated cells. Exposure to either a Src inhibitor or ISF significantly reduced the migration of cells in a transwell chamber and their ability to invade through a Matrigel-coated 8 micron-pore membrane towards a chemoattractant (media containing 10% FBS). Dasatinib produced much greater effects, and at lower concentrations, than saracatinib. When combined with ISF, the effects were enhanced, particularly with dasatinib. Conclusions: In vitro studies suggested that combining a Src inhibitor and ISF resulted in greater inhibition of metastatic potential than either alone. This may indicate that including ISF in treatment regimens may allow a lower dose of the targeting agent to be used to achieve optimal response and also decrease toxicity. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Effects of Src inhibitors and soy isoflavones on human prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2015-777

Abstract 3198: The impact of aerobic exercise on oxygenation and vascularity in breast cancer models

Background: Intratumoral hypoxia (pO2 Purpose: The goal of the present investigations was to determine whether aerobic exercise could be applied as a means to improve tumor physiology and decrease tumor hypoxia in breast cancer models. Methods: The effects of single and repeated bouts of moderate intensity exercise (treadmill running) were studied in mice bearing syngeneic murine mammary carcinoma models (EMT6 and 4T1). The exercise intensities were determined by measuring the anaerobic threshold, which is reflected by the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and once the tumors reached a size of 250 mm3, the mice were exposed to either a single bout of exercise or daily exercise bouts performed for 5 consecutive days. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. Following cessation of each exercise regime tumors were harvested, sectioned and evaluated by immunofluorescence. Vessel density (MECA-32), blood perfusion (Hoechst-33342) and hypoxia (EF5) were quantified and significance was established at p Results: Preliminary data indicate that even a single exercise bout may begin to demonstrate improvements in blood vessel perfusion and tumor oxygenation. Importantly, after 5 daily bouts of exercise, there was a significant increase in the number of patent vessels and improvement in oxygenation in the tumors of exercising mice compared to those of sedentary controls. Conclusion: The results indicate that moderate aerobic exercise can improve the oxygenation status of solid tumors. These findings suggest that such a non-toxic intervention may have potential utility in overcoming hypoxia-associated treatment resistance in cancer patients and improve patient outcome. Citation Format: Jennifer M. Wiggins, Jennifer A. Lee, Lori Rice, Dietmar Siemann. The impact of aerobic exercise on oxygenation and vascularity in breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2015-3198