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Supplemental Figure 1-2. Supplemental Figure 1: Pharmacology of POL5551; Supplemental Figure 2: Correlation of CXCR4 expression in primary tumor with survival in patients

Flow cytometric analysis of integrin β3 staining on breast cancer cells.

Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

Supplemental Figure 5. Eribulin dose ranging study.

Supplemental Figure 3. Gaussia luciferase complementation imaging model and scratch migration assay with AMD3100.

Supplemental Figure 4. MDA-MB-231 primary tumor weight and liver metastases data.

Supplemental Figure 6. Mobilization of leukocytes with POL5551.

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow–disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a “chemotherapy framing” dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. Mol Cancer Ther; 14(11); 2473–85. ©2015 AACR.

Additional details for methods and materials.

Supplemental Table 1-3. Pharmacology of POL5551 and patient characteristics at baseline

Supplemental Figure 7. Effect of POL5551 combined with eribulin on hematopoietic cells

Supplemental Figure Legends

Integrin avb3-­targeting ligand and micelle nanoparticle localization within tumor-­free bone

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. Cancer Res; 77(22); 6299–312. ©2017 AACR.

Integrin beta3 antagonist treatment on tumor cells and BMMs in culture

Anti-CSF1 treatment on β3KOM mice

Blood vessel density analysis of tumor tissue

Supplementary methods and figure legends

Flow cytometric analysis of leukocyte infiltration in tumor tissue

Antibody list for FACS

The differentially expressed genes (DEG) lists

STAT1 and STAT6 downstream gene expression

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8+ T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8+ T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484–95. ©2016 AACR.

LPS treatment enhances integrin beta3 ligand binding

Primers for qPCR

The percentage of infiltrating immune cells in B16 tumor tissue

Integrin beta3 antagonist treatment on early stage breast tumor growth

Integrin beta3 regulates STAT6 signaling

Integrin gene expression of WT and Itgb3-/- BMMs after M1 or M2 polarization

Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

// Alison K. Esser 1,* , Daniel A. Rauch 1,* , Jingyu Xiang 1 , John C. Harding 1 , Nicole A. Kohart 2 , Michael H. Ross 1 , Xinming Su 1 , Kevin Wu 1 , Devra Huey 2 , Yalin Xu 1 , Kiran Vij 1 , Patrick L. Green 2 , Thomas J. Rosol 2 , Stefan Niewiesk 2 , Lee Ratner 1 and Katherine N. Weilbaecher 1 1 Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA 2 Department of Veterinary Biosciences, School of Veterinary Medicine, The Ohio State University, Columbus, OH, USA * Co-authors Correspondence to: Katherine N. Weilbaecher, email: // Keywords : HTLV-1, ATL, leukemia, HBZ, bone Received : June 10, 2017 Accepted : August 03, 2017 Published : August 27, 2017 Abstract Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1-infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos-dependent manner. Treatment of HTLV-1-infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8+ T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8+ T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484–95. ©2016 AACR.

Fumagillin, an unstable anti-angiogenesis mycotoxin, was synthesized into a stable lipase-labile prodrug and incorporated into integrin-targeted lipid-encapsulated nanoparticles (αvβ3-Fum-PD NP). Dual anti-angiogenic therapy combining αvβ3-Fum-PD NP with zoledronic acid (ZA), a long-acting osteoclast inhibitor with proposed anti-angiogenic effects, was evaluated. In vitro, αvβ3-Fum-PD NP reduced (P

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow–disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a “chemotherapy framing” dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. Mol Cancer Ther; 14(11); 2473–85. ©2015 AACR.

We have previously demonstrated that expression of the integrin β3 (β3) subunit is consistently increased in breast cancer (BC) bone metastases across clinical samples and preclinical models. Bone metastases frequently exhibit resistance to chemotherapy. Based on evidence that integrin β3 can function as a promoter of tumor survival and metastasis, we asked whether increased β3 signaling in bone metastases promotes chemoresistance. In established murine bone metastases, the proportion of integrin β3+ tumor cells was increased after docetaxel administration. Genetic deletion of β3 in either MMTV-PyMT-derived BC cells (BO1-FL-GFP, modeling luminal B disease) or 4T1 BC cells (4T1-FL-GFP, modeling triple-negative disease) yielded bone metastases with increased sensitivity to docetaxel treatment in vivo. Retroviral rescue of β3 expression in β3-/- BO1-FL-GFP cells by a signaling-competent human integrin β3 construct (hβ3) restored relative docetaxel chemoresistance in bone metastases. By contrast, expression of a signaling-deficient mutant β3 (Δβ3) failed to restore chemoresistance, suggesting that signaling through β3 is critical to its capacity to promote docetaxel chemoresistance in bone-residing BC cells. Mechanistically, RNAseq analysis of docetaxel response in β3-/- and hβ3-rescued cells revealed β3-mediated enrichment in transcriptional pathways associated with oxidative phosphorylation and metabolism. Direct imaging of mitochondria by super-resolution microscopy and extracellular flux analysis of oxygen consumption rate further confirmed an alternative metabolic response to docetaxel in resistant, β3-expressing tumor cells. mTORC1 plays an important role in mitochondrial biogenesis and cellular metabolism, and can be activated downstream of integrin signaling in certain contexts. Based on transcriptomic data identifying enhanced mTORC1 activity as a possible mediator of β3-dependent metabolic changes, we asked whether mTOR inhibition could restore chemosensitivity in BC bone metastases. In mice bearing chemoresistant, β3-WT metastases, combination of the mTORC1 inhibitor rapamycin with docetaxel yielded synergistic, site-specific attenuation of bone tumor burden. Taken together, our data 1) establish integrin β3 as a mediator of chemoresistance in breast cancer bone metastases, 2) demonstrate a mechanistic link between integrin β3 expression and an alternative metabolic response to docetaxel in resistant cells, and 3) suggest mTORC1 inhibition as a candidate for rational combination with chemotherapy to interrupt treatment resistance in breast cancer bone metastases. Citation Format: Gregory C. Fox, Michael H. Ross, Xinming Su, Yalin Xu, Alison Esser, Elizabeth Cordell, Elizabeth Wilson, Barbara Muz, Ha Dang, Christopher A. Maher, Abdel Kareem Azab, Deborah Veis, Samuel Achilefu, Gregory M. Lanza, Katherine N. Weilbaecher. Integrin beta-3 signaling links chemoresistance and mitochondrial metabolism in breast cancer bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2613.

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. Cancer Res; 77(22); 6299–312. ©2017 AACR.

BACKGROUND Dystroglycan is a cell-surface receptor for extracellular matrix proteins including laminins and perlecan. Prior studies have shown its involvement in assembly and/or maintenance of basement membrane structures, cell polarity and tissue morphogenesis; and its expression is often reduced in prostate and other cancers. However, the role of dystroglycan in normal epithelial tissues such as the prostate is unclear. METHODS To investigate this, we disrupted dystroglycan expression in the prostate via a conditional gene targeting strategy utilizing Cre recombinase expressed in luminal prostate epithelial cells. RESULTS Contrary to expectations, deletion of dystroglycan in luminal epithelial cells resulted in no discernable phenotype as judged by histology, basement membrane ultrastructure, localization of dystroglycan ligands, cell polarity or regenerative capacity of the prostate following castration. Dystroglycan expression remains in keratin-5-positive basal cells located in the proximal ducts where dystroglycan expression is elevated in regenerating prostates. CONCLUSIONS Our results show that dystroglycan in luminal epithelial cells is not required for the maintenance of basement membranes, cell polarity or prostate regeneration. However, it is possible that persistent dystroglycan expression in the basal cell compartment may support these or other functions. Prostate 70: 777–787, 2010. © 2010 Wiley-Liss, Inc.

Dystroglycan (DG) is a cell surface receptor for extracellular matrix proteins and is involved in cell polarity, matrix organization, and mechanical stability of tissues. Previous studies documented loss of DG protein expression and glycosylation in a variety of cancer types, but the underlying mechanisms and the functional consequences with respect to cancer progression remain unclear. Here, we show that the level of expression of the βDG subunit as well as the glycosylation status of the αDG subunit inversely correlate with the Gleason scores of prostate cancers; furthermore, we show that the functional glycosylation of αDG is substantially reduced in prostate cancer metastases. Additionally, we demonstrate that LARGE2 (GYLTL1B), a gene not previously implicated in cancer, regulates functional αDG glycosylation in prostate cancer cell lines; knockdown of LARGE2 resulted in hypoglycosylation of αDG and loss of its ability to bind laminin-111 while overexpression restored ligand binding and diminished growth and migration of an aggressive prostate cancer cell line. Finally, our analysis of LARGE2 expression in human cancer specimens reveals that LARGE2 is significantly down-regulated in the context of prostate cancer, and that its reduction correlates with disease progression. Our results describe a novel molecular mechanism to account for the commonly observed hypoglycosylation of αDG in prostate cancer. Background: Dystroglycan function is decreased in prostate and other cancers. Results: Decreased functional glycosylation of α-dystroglycan is clinically correlated with reduced LARGE2 and restoring its expression rescues dystroglycan function, reducing the invasive and proliferative potential of prostate cancer cells. Conclusion: LARGE2 regulates α-dystroglycan function in the prostatic epithelium. Significance: Loss of LARGE2 expression may contribute to prostate cancer progression.

Adult T-cell leukemia/lymphoma (ATL) develops in a subset of patients infected with the HTLV-1 virus. Most ATL patients become refractory to chemotherapy and have a median survival time of 6 months. Although uncommon in hematologic malignancies, 80% of ATL patients develop osteolytic lesions and hypercalcemia of malignancy. Bone resident and metastatic tumors release paracrine factors that modulate the bone microenvironment to facilitate disease progression and decrease survival. HTLV-1 encodes 2 viral oncogenes, Tax and Hbz. Tax is critical to ATL development and regulates tumor growth and proliferation in part through trans-activation of NFκB and CREB. We have previously shown Tax expression driven by the Granzymbe B promoter is sufficient for the development of leukemia/lymphoma with osteolytic lesions and hypercalcemia. We and others have shown that Tax alters the expression of paracrine factors that modulate the bone microenvironment through effects on bone forming osteoblasts (OB) and bone resorping osteoclasts (OC). Tax is expressed in early lymphocyte transformation with low expression in advanced ATL. HBZ is expressed early in lymphocyte transformation and throughout ATL progression. We hypothesize that in ATL cells, HTLV-1 viral oncogenes Tax and Hbz cooperate to modulate bone metabolism in a paracrine manner to enhance ATL tumor growth and progression. Mice with Granzyme B driven Hbz expression (T and NK cells) develop leukemia/lymphoproliferative disease in lymph nodes correlating with increased spleen weight. We found that lymphoproliferative disease is also present in the bone marrow. Hbz mice have decreased trabecular bone at 18 months by microCT and radiographic analysis. These data suggest Hbz can alter bone metabolism. Future studies will define the effects of Hbz on bone formation, OB and OC specific effects and tumor progression. Understanding HTLV-1 oncogene modulation of the bone microenvironment will uncover critical pathways in tumor/bone cross talk enabling the development of novel targeted therapies for ATL patients. Citation Format: Alison Esser, Dan Rauch, Jingyu Xiang, John Harding, Nicole Kohart, Patrick Green, Stefan Niewiesk, Thomas Rosol, Lee Ratner, Katherine Weilbaecher. HTLV-1 viral oncogene Hbz induces leukemia with osteolytic bone involvement in mice. [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 3285.

Background: Bone metastases occur in 70% of metastatic breast cancer patients and are a leading cause of morbidity. Current therapies are often palliative, in part due to a lack of specificity for tumor targets within the bone. Integrin αvβ3 is overexpressed on neo-angiogenic blood vessels, tumor-promoting macrophages, osteoclasts, and more aggressive breast cancer cells, making it an attractive therapeutic target. Objective: The goal of this study was to use Sn2 lipase-labile docetaxel-prodrug nanoparticle, targeted against activated integrin αvβ3, to attenuate breast cancer metastases. Methods: A novel phospholipid-based micelle (∼12.5 nm) was functionalized with a peptidomimetic for activated integrin αvβ3, and designed to carry either rhodamine for fluorescent labeling or Sn2 lipase-labile prodrug of docetaxel (DTX-PD) for drug delivery. For microscopic localization studies, fluorescently labeled micelles were prepared with or without integrin αvβ3-targeting. C57BL/6 female mice received MMTV-PyMT breast cancer cell line (luciferase-labeled) via intracardiac (IC) injection to achieve tumor metastasis in all major organs. On day 8 post-IC injection, micelle preparations were administered i.v. and circulated within C57BL/6 mice for 3 hours prior to sacrifice and tissue collection. For drug efficacy studies, Sn2 lipase-labile docetaxel-prodrug was incorporated into αvβ3-micelles (αvβ3-DTX-PD). C57BL/6 female mice IC injected with MMTV-PyMT cells (luciferase-labeled) were treated with αvβ3-DTX-PD, or molar equivalent dose of free-DTX, or saline. Beginning on day 4 post-IC injection, mice were treated 3 times, once every 3 days (1.85mg/kg DTX per treatment). On day 12 post-IC injection, metastatic burden in the major organs was analyzed via ex vivo bioluminescent imaging. Results: Fluorescent histological analysis of the tibiofemoral bone region showed significant colocalization of αvβ3-micelles with breast cancer bone metastases, as compared with non-targeted micelles (6.5-fold increase, p Conclusion: These findings suggest that the unique elevated expression of integrin αvβ3 within breast cancer bone metastases could be exploited with αvβ3-DTX-PD micelles for effective therapy. Citation Format: Michael H. Ross, Alison K. Esser, Anne H. Schmieder, Grace Cui, Xiaoxia Yang, Xinming Su, Dipanjan Pan, Gregory M. Lanza, Katherine N. Weilbaecher. Integrin αvβ3-targeted lipase-labile docetaxel-prodrug micelles preferentially treat breast cancer bone metastases. [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 2198.

The vasoactive peptide endothelin-1 (ET-1) has been implicated in promoting the progression of prostate and other cancers though its precise mechanism(s)-of-action remain unclear. To better define the role of ET-1 in prostate cancer progression, we generated prostate cancer cell lines (PC-3 and 22Rv1) that express elevated levels of ET-1. As anticipated, increased ET-1 lead to modest autocrine growth stimulation of PC-3 cells in monolayer culture and increased colony formation in soft agar by both cell lines. Unexpectedly, however, metastatic colonization of 22Rv1 cells expressing elevated levels of ET-1 was reduced, as was the size of subcutaneous tumors produced by both 22Rv1- and PC-3 cells. Based on these data, we hypothesized that high levels of ET-1 may negatively impact the tumor microenvironment. We found that increased ET-1 expression did not consistently inhibit angiogenesis, indicating that this was not the cause of poor tumor growth. As an alternative explanation, we examined whether elevated ET-1 results in local vasoconstriction and thus reduces the blood supply available to the tumor. Consistent with this hypothesis, treatment of mice bearing PC-3 xenografts with a vasodilator increased tumor perfusion and partially restored tumor growth. Moreover, analysis of tumor vascular casts indicated vasoconstriction of tumor-feeding arterioles. Taken together, our data suggest that the local concentration of the ET-1 peptide is critical for determining a balance between its previously unrecognized tumor growth-suppressing activity (vasoconstriction) and known growth-promoting (mitogenesis, survival and angiogenesis) activities. These findings may have implications for the modification of current prostate cancer therapies involving ET-1.

Cancer cells exhibit altered interactions with their extracellular matrix microenvironment that are thought to underlie tumor progression and metastasis. Dystroglycan (DG) is a receptor for extracellular matrix proteins including laminins and perlecan that consists of a ligand-binding α subunit and a transmembrane β subunit. DG protein expression is reduced in numerous types of cancer, including prostate cancer. Typically DG mRNA expression is not remarkably altered and mutations in the DG gene have not been reported in any cancer to date. We have investigated the basis for reduced DG expression in prostate cancer. Using tissue microarray (n=50; Gleason score 6-8) we show that loss of either α or β DG independently predicts Gleason score. We show that loss of αDG immunostaining in human prostatectomy samples (n=138) trended towards predicting overall mortality independent of grade and stage with an estimated hazard ratio of 1.32 (CI=0.92, 1.89; p=0.1381). Furthermore, analysis of prostate cancer metastases in bone, lung and lymph node shows a near universal loss of αDG immunostaining. Importantly, we observed cases in which αDG is reduced while βDG persisted in serial sections, suggesting that these subunits may be differentially affected. In these studies, we detected αDG using a glycosylation-sensitive antibody IIH6. Therefore, we investigated the basis of altered αDG glycosylation using a series of prostate cancer cell lines. We found that αDG glycosylation is heterogeneous across several prostate cancer cell lines. Importantly, we found that an aggressive derivative of PC-3 cells lacks IIH6 immunoreactivity, but not core αDG protein expression; whereas the bulk PC-3 population is IIH6 positive. We evaluated the relative expression of a panel of glycosyltransferases known to modify αDG in these cell lines and found that loss of IIH6 immunoreactivity is correlated with loss of LARGE2 expression. This result contrasts with previous findings implicating LARGE or iGNT with abnormal glycosylation of αDG in breast and prostate cancer respectively. Reduced expression of LARGE2 is also observed in pancreatic cancer cell lines and is correlated with loss of IIH6 immunoreactivity. We show that LARGE2 is involved in the functional glycosylation of αDG in prostate cancer cells. shRNA-mediated knockdown of LARGE2 in prostate cancer cells does not affect growth rate, but does increase transwell migration across laminin-1 substrate. Our results are the first to implicate loss of LARGE2 expression as a means to explain the loss of appropriate glycosylation of αDG in prostate or other cancers, and indicates that LARGE2 function may modulate prostate cancer progression and metastasis. 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 439. doi:1538-7445.AM2012-439

Dystroglycan, an extracellular matrix receptor, is expressed in many tissues including muscle, neural, adipose and epithelial where its function includes roles in polarity, basement membrane formation and tissue morphogenesis. Loss of dystroglycan expression has been shown in many epithelial cancers including prostate cancer yet the underlying mechanism remains unclear. Here, we investigate the role of DG in prostate cancer progression. We show that staining for the extracellular αDG subunit with the glycosylation-sensitive IIH6 anti-αDG antibody as well as the transmembrane βDG subunit are independent predictors of Gleason score in clinical prostate cancer specimens. In addition, increased αDG glycosylation due to expression of LARGE, a putative glycosyltransferase, in prostate cancer cell lines results in decreased tumor growth in prostate cancer xenografts. We determined that this reduction in growth is specific to the effect of LARGE on dystroglycan. Therefore our work has shown that the extent of αDG glycosylation predicts Gleason scores in prostate cancer and that LARGE dependent glycosylation of αDG regulates tumor growth. This work is supported by a grant from the National Institutes of Health R01-CA130916 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 421.