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Supplemental Methods, References, and Figure Legends

Vinculin is localized to cell-ECM borders in invasive breast cancer and adhesome proteins are upregulated across human cancers.

Scanning angle interference microscopy - validation experiments.

MECs remodel 3D collagen gels for cell invasion. Individual collagen fibers are stiff and >500nm in diameter.

Vinculin nanoscale localization reflects altered molecular activity.

List of Adhesome, Oncogene and Control Genes used in Bioinformatics Analysis.

ECM cues, malignant transformation, and integrin signaling, promote cell spreading and invasion.

List of Cancer Tissue with Unbiased Matched Normal Tissue used in Bioinformatics Analysis.

The prognosis of patients with metastatic and recurrent osteosarcoma has not improved over the last 30 years because no effective treatment strategy has been established for lung metastases. Although molecular-targeted drugs that modify the extracellular environment, such as antifibrotic agents, have been developed for cancer treatment, the suppressive effects of antifibrotic agents on osteosarcoma lung metastasis are unclear. Osteosarcomas need to adapt to considerable changes with respect to the stiffness of the environment and fibrosis during lung metastasis and may thus be vulnerable to fibrotic suppression as they originate at the site of a stiff bone with considerable fibrosis. In our study, we investigated whether fibrosis was a therapeutic target for suppressing osteosarcoma metastasis. Lung tissue samples from patients and a mouse model (LM8-Dunn model) showed that lung metastatic colonization of osteosarcoma cells proceeded with massive lung fibrosis. Metastatic osteosarcoma LM8 cells proliferated in a scaffold-dependent manner; the proliferation was less dependent on YAP-mediated mechanotransduction on soft polyacrylamide gels. The antifibrotic agents pirfenidone and nintedanib suppressed lung metastasis in the LM8-Dunn model. The osteosarcoma cells did not show increased proliferation, as reported in breast cancer, after continuous culture in a soft environment. We speculated that the antifibrotic agents were effective because the osteosarcoma cells remained scaffold-dependent in the soft tissue environment. Thus, antifibrotic strategies may be useful in suppressing lung metastasis of bone and soft tissue tumors with stiff primary sites such as those in osteosarcoma.

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression. Tissue-specific factors linking stem-progenitor cell frequency to cancer risk and progression remain ill defined. Using a genetically engineered mouse model that promotes integrin mechanosignaling with syngeneic manipulations, spheroid models, and patient-derived xenografts we determined that a stiff extracellular matrix and high integrin mechanosignaling increase stem-progenitor cell frequency to enhance breast tumor risk and progression. Studies revealed that high integrin-mechanosignaling expands breast epithelial stem-progenitor cell number by potentiating progesterone receptor-dependent RANK signaling. Consistently, we observed that the stiff breast tissue from women with high mammographic density, who exhibit an increased lifetime risk for breast cancer, also have elevated RANK signaling and a high frequency of stem-progenitor epithelial cells. The findings link tissue fibrosis and integrin mechanosignaling to stem-progenitor cell frequency and causally implicate hormone signaling in this phenotype. Accordingly, inhibiting RANK signaling could temper the tumor promoting impact of fibrosis on breast cancer and reduce the elevated breast cancer risk exhibited by women with high mammographic density.SummaryElevated mechano-signaling and matrix stiffness promote progesterone and RANK mediated expansion of mammary progenitors and breast cancer risk and progression.

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression. Tissue-specific factors linking stem-progenitor cell frequency to cancer risk and progression remain ill defined. Using a genetically engineered mouse model that promotes integrin mechanosignaling with syngeneic manipulations, spheroid models, and patient-derived xenografts we determined that a stiff extracellular matrix and high integrin mechanosignaling increase stem-progenitor cell frequency to enhance breast tumor risk and progression. Studies revealed that high integrin-mechanosignaling expands breast epithelial stem-progenitor cell number by potentiating progesterone receptor-dependent RANK signaling. Consistently, we observed that the stiff breast tissue from women with high mammographic density, who exhibit an increased lifetime risk for breast cancer, also have elevated RANK signaling and a high frequency of stem-progenitor epithelial cells. The findings link tissue fibrosis and integrin mechanosignaling to stem-progenitor cell frequency and causally implicate hormone signaling in this phenotype. Accordingly, inhibiting RANK signaling could temper the tumor promoting impact of fibrosis on breast cancer and reduce the elevated breast cancer risk exhibited by women with high mammographic density. Citation Format: Jason J. Northey, Yoshihiro Yui, Mary-Kate Hayward, Connor Stashko, FuiBoon Kai, Janna Mouw, Dhruv Thakar, Jonathon Lakins, Alastair Ironside, Susan Samson, Rita Mukhtar, E. Shelley Hwang, Valerie Weaver. Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB021.

Mechanical stimulation by the extracellular matrix (ECM) controls physiological and pathological cellular responses, such as stem cell differentiation, organogenesis, and tumor progression. Polyacrylamide (PA) gels have been widely used to study cell-ECM mechanical interactions. Typically, sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-SANPAH) is used as a protein crosslinker in these gels. However, its low solubility, unstable binding with proteins, and high cost are barriers to its application. The objective of this study was to improve and simplify the preparation of PA gels using an economical crosslinker, N-hydroxysuccinimide-acrylamide (NHS-AA) ester, to enable increased stability in protein coating. By exposing excess NHS to the gel surface, we found an optimal ratio of NHS-AA ester:AA to obtain NHS-AA ester-containing PA gels with a uniform ECM protein coating and stiffness similar to that of sulfo-SANPAH-containing PA gels. The biological behavior of MCF7 and MCF10A cells were similar on NHS-AA ester and sulfo-SANPAH gels. Acini formation in Matrigel overlay culture were also consistent on NHS-AA ester and sulfo-SANPAH gels. This novel PA gel preparation method using NHS-AA ester can effectively replace the sulfo-SANPAH method and will be immensely useful in the evaluation of cell-ECM mechanical interactions.

Background Clinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells. Methods The molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment. Results Changes in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner. Conclusion These findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

The prognosis for leptomeningeal metastasis (LM) remains extremely poor regardless of intrathecal chemotherapy with various drugs, and thus, new treatments are necessary. Butyrate is an endogenous 4-carbon saturated fatty acid, has been investigated as an anti-tumor agent because of its multiple suppressive effects on several tumors. In this study, we investigated the cellular basis of sodium butyrate (SB), a sodium salt compound of butyrate, in vitro and evaluated the clinical potential of intrathecal SB administration for LM in vivo. We examined SB’s effects on Walker 256 rat mammary tumor cells with regard to cytotoxicity, cell morphology, colony formation, migration, and invasion. We also examined SB’s neurotoxicity for primary neurons and primary astrocytes. We finally evaluated the potency of continuous intrathecal SB administration in rats with intrathecally transplanted breast tumors as an LM model. Physiological SB concentrations (2–4 mM) induced growth suppression, morphological changes, and inhibition of migration and invasion, but did not exhibit neurotoxic effects on primary neurons and astrocytes. Continuous intrathecal SB administration in a rat LM model significantly increased survival periods with little neurotoxicity. Continuous intrathecal SB administration significantly improved prognoses in a rat LM model, which suggests that SB is a promising therapy for LM.

Lung metastasis markedly reduces the prognosis of osteosarcoma. Moreover, there is no effective treatment for lung metastasis, and a new treatment strategy for the treatment of osteosarcoma lung metastasis is required. Therefore, in this study, we investigated the suppressive effect of the microtubule inhibitor eribulin mesylate (eribulin) on lung metastasis of osteosarcoma. At concentrations >proliferation IC50, eribulin induced cell cycle arrest and apoptosis in a metastatic osteosarcoma cell line, LM8. However, at concentrations

Extracellular matrix (ECM) stiffness induces focal adhesion assembly to drive malignant transformation and tumor metastasis. Nevertheless, how force alters focal adhesions to promote tumor progression remains unclear. Here, we explored the role of the focal adhesion protein vinculin, a force-activated mechanotransducer, in mammary epithelial tissue transformation and invasion. We found that ECM stiffness stabilizes the assembly of a vinculin–talin–actin scaffolding complex that facilitates PI3K-mediated phosphatidylinositol (3,4,5)-triphosphate phosphorylation. Using defined two- and three-dimensional matrices, a mouse model of mammary tumorigenesis with vinculin mutants, and a novel super resolution imaging approach, we established that ECM stiffness, per se, promotes the malignant progression of a mammary epithelium by activating and stabilizing vinculin and enhancing Akt signaling at focal adhesions. Our studies also revealed that vinculin strongly colocalizes with activated Akt at the invasive border of human breast tumors, where the ECM is stiffest, and we detected elevated mechanosignaling. Thus, ECM stiffness could induce tumor progression by promoting the assembly of signaling scaffolds, a conclusion underscored by the significant association we observed between highly expressed focal adhesion plaque proteins and malignant transformation across multiple types of solid cancer. See all articles in this Cancer Research section, “Physics in Cancer Research.” Cancer Res; 74(17); 4597–611. ©2014 AACR.

Osteosarcoma is the most common malignant bone tumor among children and adolescents. The prognosis of osteosarcoma remains poor in these thirty years because the effective therapeutic option for lung metastasis has not been established. Therefore, the development of effective metastatic therapy is required. Eribulin, a new microtubule targeting agent, has been indicated for recurrent breast cancer and malignant soft tissue sarcoma. Clinical trials of eribulin showed that it elongated overall survival period rather than disease-free survival period. These results motivated us to investigate the effect of eribulin on metastasis suppression of osteosarcoma. Here, we examined the inhibitory effect of eribulin on lung metastasis and its mechanism using murine osteosarcoma metastasis model. In vivo experiments: Eribulin was intravenously injected by two administration methods, (1) high dose administration on standard schedule; 1 mg/kg q7d × 2, (2) frequent low dose administration; 0.3 mg/kg q4d × 4. Both methods sufficiently reduced circulating tumor cells in blood and inhibited lung metastasis. Method (1) also inhibited primary tumor growth, but induced severe body weight loss. Method (2) had little effect on primary tumor growth and showed little body weight loss. In vitro experiments: The IC50 concentration of eribulin for metastatic osteosarcoma cell line, LM8, was about 20 nM. Pharmacokinetics studies have shown that eribulin intravenously administered at 1 mg/kg presents with a brief high-concentration phase which surges to ≥100 nM followed by a long low-concentration phase which stabilizes at ~10 nM for one week. These two phases sandwiched the IC50 concentration of eribulin. Thus, we separately investigated that the effect of high and low eribulin concentrations on LM8. High eribulin concentrations induced cell cycle arrest and apoptosis in LM8, whereas low eribulin concentrations changed cell morphology and decreased cell migration through reducing directionality and focal adhesion turnover. Reduced peripheral localization of adenomatous polyposis coli (APC) protein by eribulin might cause these effects. In a three-dimensional collagen culture system, low eribulin concentrations inhibited tumor cell infiltration and colony formation. The anti-metastatic effects at low concentrations backed-up the results of our in vivo experiments by frequent low dose administration method. Eribulin is a potential therapeutic option for lung metastasis of osteosarcoma. Frequent low dose administration method with fewer side effects enables the combination with other chemotherapeutic agents and broadens the application of eribulin as an anti-metastatic drug for osteosarcoma. Citation Format: Kenta Watanabe, Yoshihiro Yui, Satoru Sasagawa, Kayo Suzuki, Masahiko Kanamori, Taketoshi Yasuda, Tomoatsu Kimura. Low-dose eribulin suppresses lung metastasis of osteosarcoma in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1995.

The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin β1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin β1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury.

Malignant rhabdoid tumors (MRT) exhibit a very poor prognosis because of their resistance to chemotherapeutic agents and new therapies are needed for the treatment of this cancer. Here, we show that the histone deacetylase (HDAC) inhibitor FK228 (depsipeptide) has an antitumor effect on MRT cells both in vitro and in vivo. FK228 is a unique cyclic peptide and is among the most potent inhibitors of both Class I and Class II HDACs. FK228 inhibited proliferation and induced apoptosis in all MRT cell lines tested. Preincubation with the pancaspase inhibitor zVAD-fmk did not completely rescue FK228-induced cell death, although it did inhibit apoptosis. Transmission electron microscopy (TEM) showed that FK228 could stimulate MRT cells to undergo apoptosis, necrosis or autophagy. FK228 converted unconjugated microtubule-associated protein light chain 3 (LC3-I) to conjugated light chain 3 (LC3-II) and induced localization of LC3 to autophagosomes. Apoptosis inducing factor (AIF), which plays a role in caspase-independent cell death, translocated to the nucleus in response to FK228 treatment. Moreover, small interfering RNA (siRNA) targeting of AIF prevented the morphological changes associated with autophagy and redistribution of LC3 to autophagosomes. Disrupting autophagy with chloroquine treatment enhanced FK228-induced cell death. In vivo, FK228 caused a reduction in tumor size and induced autophagy in tumor tissues. Using immunoelectron microscopy, we confirmed AIF translocation into the nucleus of FK228-induced autophagic cells in vivo. Thus, FK228 is a novel candidate for an antitumor agent for MRT cells.

The histone deacetylase inhibitor depsipeptide \[(1 S ,4 S ,7 Z ,10 S , 16 E ,21 R )-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone\] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC50) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers. © 2014 Nature America, Inc. All rights reserved.

Tissue stiffness has emerged as an important regulator in the maintenance of normal cellular processes. In general, solid tumors are known to be much stiffer than normal tissue, with increased extracellular matrix (ECM) density being indicative of a higher risk for many cancers, including those of the breast. Over the course of breast cancer progression, the ECM becomes increasingly stiffer, a phenomenon which is known to be a driver of malignancy. In a 3D culture system using self-assembling peptide (SAP) gels, we grew MCF10A acini under a dynamic range of stiffness environments (140-5800 Pa). Under normal breast tissue elasticity, mammary epithelial cells form rounded, polarized acini with cleared lumens. However, in tumor-like stiffnesses, the same cells form non-polarized, collapsed structures that invade into the gel. To investigate the specific genes and pathways affected by stiffness, we have conducted tandem mass spectrometry to identify protein expression changes in MCF10A cells grown on SAP gels of increasing stiffness. Interestingly, the resulting analysis showed that cells grown on higher stiffnesses exhibit greater variation in protein expression. These divergent force-regulated molecular pathways are involved in a variety of cellular functions, including cell adhesion and migration, metabolism, and the immune response, among others. This study underscores the significance of mechanical forces exerted by the ECM, and signifies the first large-scale proteomic screen carried out to analyze heterogeneous cellular responses to changing stiffness environments. Citation Format: Sonya Liu, Russell Bainer, Yoshihiro Yui, Colin Flinders, Parag Mallick, Valerie Weaver, Shannon Mumenthaler. Characterization of diverging molecular and phenotypic responses under changing extracellular matrix stiffness in MCF10A cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A23.

s: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WA Breast cancers frequently develop treatment resistance that leads to recurrence, dissemination and patient mortality. Among the mechanisms that foster treatment resistance is the ability of tumor cells to undergo an epithelial-to-mesenchymal transition (EMT) and exhibit stem-like behavior. Accumulating evidence now supports the concept that the mechanical properties of the extracellular matrix microenvironment can critically influence developmental cell fate and modify several features of tumor progression. Data from our laboratory using preclinical models and clinical samples suggest that tissue tension and elevated mechanosignaling increase prior to and accompany malignant transformation. Therefore, we hypothesize that enhanced tissue mechanical tension and high mechanosignaling foster mammary stemness, EMT and breast cancer aggression. We tested this prediction by generating transgenic mice conditionally expressing a β1-Integrin clustering mutant (V737N) in the mammary epithelium. V737N expression stimulated integrin-mediated mechanosignaling in mammary epithelial cells (MECs), as determined by elevated phosphorylation of FAK and p130Cas, and this heightened mechanosignaling promoted precocious epithelial ductal branching, end bud formation and increased MEC proliferation in the mammary gland. These mammary phenotypes were accompanied by an increase in the ratio of basal/myoepithelial to luminal MECs in V737N mammary glands. Isolated V737N-expressing MECs possessed higher levels of genes associated with EMT and stemness, and Matrigel colony formation and transplantation assays revealed a functional increase in progenitor/stem cell frequency in the V737N-expressing basal/myoepithelial MECs compared to their corresponding controls. To examine the effect of tissue tension on breast cancer progression, we combined the V737N-β1-Integrin together with a mouse model of HER2-positive breast cancer (MMTV-NEU). While V737N expression had no observable effect on primary tumor outgrowth, overall tumor incidence and lung metastasis were significantly augmented. Further histological examination of tumors and gene expression analysis uncovered a phenotypic shift, such that V737N tumors displayed expression patterns resembling an EMT-like basal tumor when compared to control tumors. Thus, tissue tension by way of enhanced mechanosignaling promotes mammary stemness, higher mammary tumor incidence and a more basal-like aggressive tumor character. Potential V737N mechanistic actions currently under investigation involve altered orientation of cell divisions resulting from disrupted MEC polarity, as well as a heightened sensitivity to hormone-induced expansion of stem/progenitor cells. (Supp by: USMRAA Department of Defense-BCRP BC122990 and NIH NCI R01CA192914 to VW and AACR 15-40-01-NORT to JN). Citation Format: Jason J. Northey, Yoshihiro Yui, Janna K. Mouw, Johnathan N. Lakins, Valerie M. Weaver. Tissue tension promotes mammary stemness and breast cancer aggression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR04.

High BAALC (brain and acute leukemia, cytoplasmic) gene expression may indicate an adverse prognosis for adults who have acute myeloid leukemia (AML) and a normal karyotype, but its prognostic significance for pediatric AML cases is unclear. Whether different BAALC isoform patterns are of prognostic significance is also unclear. Newly diagnosed AML patients with normal karyotype who were treated by the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed in terms of their BAALC expression levels (n = 29), BAALC isoforms (n = 29), and CEBPA mutations (n = 49). Eleven and 18 patients exhibited high and low BAALC expression, respectively, but these groups did not differ significantly in terms of overall survival (54.6 vs. 61.1%, P = 0.55) or event-free survival (61.4 vs. 50.0%, P = 0.82). Three of these 29 patients (10.3%) expressed the exon 1-5-6-8 BAALC isoform along with the expected 1-6-8 isoform and had adverse clinical outcomes. Novel CEBPA mutations were also identified in four of 49 patients (8.2%). All four patients have maintained complete remission for at least 5 years. Thus, 1-5-6-8 isoform expression may be associated with an adverse prognosis in pediatric AML with normal karyotype. CEBPA mutations may indicate a favorable prognosis.

A 6-year-old male with chronic granulomatous disease, who was transplanted with bone marrow and exhibited increasing mixed chimerism, subsequently received two donor lymphocyte infusions (DLI). Two weeks after the second DLI, the patient developed acute graft-versus-host disease (GVHD) and progressive pancytopenia that was associated with autoantibody production. Conventional treatment did not improve the pancytopenia. However, administration of Rituximab (RTX) (375 mg/m2/week for four consecutive weeks) resulted in a rapid resolution of the pancytopenia. The patient achieved full donor chimerism without GVHD symptoms. RTX can be valuable for managing immune-mediated cytopenias that arise after DLI and are refractory to conventional therapies. Pediatr Blood Cancer 2010;54:329–331. © 2009 Wiley-Liss, Inc.

The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC(50)) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

A four-yr-old boy developed a solitary metastasis nine months after living-related liver transplantation for unresectable hepatoblastoma. After resection of the metastatic lesion, he received an auto-PBSCT with a double-conditioning regimen consisting of melphalan and thiotepa. Auto-PBSCT could be safely performed without any serious regimen-related toxicity or infection. However, transient cessation of tacrolimus during myelosuppression resulted in graft rejection of the liver just after hematological engraftment, but rejection was resolved by tacrolimus and methylprednisolone. The patient is alive and free from disease two yr after auto-PBSCT without any signs of graft rejection. High-dose chemotherapy using this conditioning regimen may be feasible for recurrent hepatoblastoma after liver transplantation in terms of safety and anti-tumor activity.

We performed an intravenous cyclosporine (CsA) drip infusion method for 3 hours and C3 monitoring in pediatric hematopoietic stem cell transplantation and examined the internal change and monitoring method of CsA. A total of six cases comprised five cord blood transplantations and one related allogeneic bone marrow transplantation. We started CsA 1.5 mg/kg at the day before transplantation by intravenous drip infusion (twice a day) for three hours. We controlled the dose so that the optimal peak value of C3 reached 800-1000 ng/ml. We recognized the C3 peak occurred three hours after initiation of infusion, and the blood CsA concentration was restored to the baseline value (C0) to (C12) 12 hours after that. We found a strong correlation between the C3 value (r = 0.90), and AUC(0-12). Two patients had grade II acute graft-versus-host disease (GVHD), but one needed no treatment, and the other recovered with short-term dosage of prednisolone. Apart from these instances, no serious complication occurred. In pediatric hematopoietic stem cell transplantation, it seems that regulation of the appropriate blood CsA concentration is enabled by using C3 monitoring at around 3 hours after commencing the intravenous drip infusion method for CsA.

The stiffness of the extracellular matrix (ECM) drives mechanosignaling that regulates tissue development and malignancy. We previously showed that a stiff ECM disrupts tissue organization and enhances malignant progression by inducing cell invasion and migration. However, the specific transcriptional and molecular events in which mechanotransduction directs these phenotypes are not well understood. To clarify this process, we used a combination of genome-scale approaches to monitor changes in gene expression and protein abundance as a function of acinar morphogenesis and tissue homeostasis in three dimensional extracellular matrix hydrogels with tunable stiffness. Elevated ECM stiffness perturbed tissue homeostasis and reverted the transcriptional phenotype of differentiated mammary acini to resemble that observed in rapidly proliferating nonpolarized mammary cell aggregates. These findings suggest that tissue tension induces cellular changes that directly reflect higher-order tissue organizational states. We found that these changes involve the spatial rearrangement of peripheral chromatin, and that the expression levels of multiple histone deacetylases increase in organized tissues concurrently with elevated nuclear heterochromatin content, an effect that is abrogated in rigid ECM conditions. We support these observations by mapping mechanoresponsive peripheral heterochromatin elements via ChIPseq, enabling us to directly identify dynamic regions containing genes whose transcriptional activity is responsive to mechanical cues. Finally, using a combination of genomic, imaging, and molecular biology techniques we demonstrated that ECM compliance and tissue organization significantly influences global RNA abundance. Notably, this model presents formidable conceptual and practical challenges for the interpretation of genomic data. Collectively, this work indicates that tissue organization is critically dependent on the cellular mechanical environment, which qualitatively and quantitatively shapes the epigenetic and transcriptional landscape by mechanisms that have not yet been elucidated. Note: This abstract was not presented at the conference. Citation Format: Russell Bainer, Yoshihiro Yui, Shannon Mumenthaler, Parag Mallick, Lin Liu, Hua-Jun Wu, Ondrej Podlaha, Franziska Michor, Jan Liphardt, Jonathan Licht, Valerie Weaver. Extracellular stiffness cues drive spatial reorganization of the genome to globally constrain RNA abundance. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR09.

The stiffness of the extracellular matrix (ECM) stimulates mechanotransduction pathways that regulate tissue development and tumor progression. We previously showed that a stiff ECM potentiates cell growth and survival, enhances cell migration to drive tumor cell invasion, and drives malignant progression of the mammary gland in culture and in vivo, but the specific transcriptional and molecular events that occur as cells acquire these phenotypic changes are not well understood. To clarify this process, we used expression microarrays, tandem mass spectrometry, and RNA sequencing to identify changes in gene expression levels, isoform usage, and protein abundance that occur as intact ascini respond to distinct stiffness environments. We found that in stiffer ECM conditions ascini acquire consistent changes in gene expression related to cell adhesion and mRNA splicing, and specifically induce the expression of a set of genes involved in epithelial cell differentiation that includes multiple SPRR and S100 proteins. Remarkably, these genes map to an apparent stiffness-mediated transcriptional hotspot on chromosome 1q21, a region containing elevated transcription in many cancers but whose activity has not been related to mechanotransduction. We then used a heuristic approach to identify additional candidate force-mediated transcriptional hotspots throughout the genome that contain multiple genes that are coordinately activated or silenced in response to elevated ECM stiffness. Provocatively, we find that genes whose expression levels are responsive to ECM stiffness cues are disproportionately located within chromosomal regions that associate with the nuclear lamina, suggesting that these transcriptional changes may be due in part to force-dependent alteration of genomic contacts with the nuclear envelope. These studies provide biological insight into the divergent cellular responses to distinct stiffness environments and suggest that genome regulatory responses to the force environment may specifically target distinct chromosomal regions via a mechanism that remains to be elucidated. Citation Format: Russell Bainer, Yoshihiro Yui, Shannon Mumenthaler, Parag Mallick, Ondrej Podlaha, Franziska Michor, Jan Liphardt, Jonathan Licht, Valerie Weaver. 3D extracellular stiffness cues drive localized changes in gene expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2014-2344

Tumor metastasis is the major cause of morbidity and mortality from breast cancer, yet there are no effective management strategies and treatments for metastatic breast cancer. These challenges urge us to have a better understanding of tumor metastasis. Factors in the host microenvironment, including stromal cells, cytokines, and non-cellular extracellular matrix (ECM), are known playing important roles on the dynamic process of tumor metastasis. Our and other recent studies have shown breast tumor progression is associated with ECM remodeling, increased collagen cross-linking and tissue stiffening. Tissue stiffening, in particular, promotes tumor cell invasion in MMTV-Her2/Neu mouse model by enhancing the cross talk between the focal adhesion and oncogenic signaling 1. However, ECM is involved in many steps of tumor metastasis, we do not know explicitly if ECM and tissue stiffness would affect the final tumor metastasis, if so how. We answered our questions by investigating the effects of altered tissue stiffness on the lung metastasis in the MMTV-PyMT mouse model for breast cancer as MMTV-PyMT mice develop lung metastasis with 100% efficiency. We inhibited Lysyl oxidase mediated collagen I crosslinking (beta-aminopropion, BAPN 1) in MMTV-PyMT mice at different ages, measured mammary tissue stiffness with high-resolution tissue diagnostic probe, and examined the lung metastasis. We found that there was a close correlation between tissue stiffness and lung metastasis and that preventing tissue stiffening reduced lung metastasis in PyMT model without affecting primary tumor growth. In addition, we found this reduced lung metastasis was correlated with fewer circulating tumor cells. Further studies showed that BAPN inhibited tumors were less hypoxic and that the tumor cells were less migratory than their corresponding controls. These results suggest that increased collagen crosslinking and tissue stiffness contribute to breast tumor metastasis by facilitating tumor cell migration and intravasation processes. A stiffer tissue and ECM not only modulate the capability of tumor cells to migrate but also affect the substrates on which tumor cells migrate. These two major effects of tissue stiffening make tumor cells succeed in intravasation, which is required for their subsequent lung colonization. We are now investigating the underlying molecular and biomechanical mechanisms involved in this specific situation. These studies, together with our previous work on MMTV-Her2/Neu model 1, show that tissue stiffness modulates multiple stages of breast cancer metastasis and will help us to develop novel strategies of managing metastatic breast caners. (Support by DOD.WX81XWH-05-1-0330, NIH/NCI RFA-CA-09-009) 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 1477. doi:1538-7445.AM2012-1477

The extracellular matrix and its receptors regulate tissue development and homeostasis and perturbations in this dialogue contribute to the pathogenesis of cancer. In the mammary gland β1 integrin modulates branching morphogenesis and directs tissue-specific differentiation so that expression of a dominant-negative or the conditional knockdown of β1 integrin compromises mammary epithelial cell (MEC) proliferation, survival and migration to disrupt branching and acinar differentiation (Streuli et al. J Cell Biol. 1991). Consistently, breast tumors are associated with elevated β1 integrin signaling through focal adhesion kinase (FAK) and ablation of β1 integrin or FAK expression prevents tumor progression and inhibits metastasis (White et al. Cancer Cell. 2004, Pylayeva et al. J Clin Invest. 2009, Provenzano et al. Am J Pathol. 2008). Nevertheless, whether integrin-dependent signaling promotes cell proliferation, survival and migration to drive mammary epithelial branching and whether this enhanced signaling would be sufficient to drive tumorigenesis in vivo has yet to be determined. We developed a novel β1 integrin mutant V737N in which the neutral transmembrane amino acid glycine was replaced with a highly charged asparagine (Paszek et al. Cancer Cell. 2005). This integrin mutant promotes the assembly of focal adhesions and potentiates FAK and ERK activity in fibroblasts and mammary epithelial cells even when the cells interact with a compliant ECM. The mutant integrin also enhances cell survival and promotes invasion of premalignant MECs within compliant three dimensional (3D) gels and promotes tumor growth in vivo. To further explore the impact of enhanced focal adhesion assembly on tissue morphogenesis and tumorigenesis we conditionally knocked in V737N integrin mutant into the ROSA26 locus using PBigT vector. These mice were crossed into the MMTV-Cre mice to induce expression of V737N β1 integrin in the luminal MECs and effects on developmental branching morphogenesis were assessed. Data showed that the mammary glands of 10 week old virgin V737N β1 integrin mice were larger and heavier as compared to age matched controls. Whole mount analysis revealed a profound precocious branching morphogenesis in the number 4 mammary glands that was characterized by elevated numbers of tertiary branches with prominent terminal end buds. Moreover, immunohistochemistry of the mammary epithelial cells showed evidence of elevated proliferation. These findings suggest that forced assembly of integrin focal adhesions is sufficient for mammary epithelial cell proliferation and migration. Further studies are underway to explore whether enhancing focal adhesion assembly potentiates integrin and growth factor signaling and if this compromises tissue specific differentiation and/or promotes malignancy. Supported by: NCI SPORE P50 CA058207, NCI R01 CA138818-01A1, NCI R01 CA140663-01-A2, DOD W81XWH-05-1-0330 and NCI U54 CA143836-01 to VMW. 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 LB-494. doi:1538-7445.AM2012-LB-494

The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl+ cells. While they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we assessed the regulatory mechanisms for cellular survival and death of Bcr-Abl+ leukemias more precisely, using a novel Bcr-Abl tyrosine kinase inhibitor, INNO-406 (formerly NS-187) which is more selective and 25-55-fold more active than imatinib (Kimura S, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl), and in vivo CML mouse model. INNO-406 induces apoptosis in all lines examined, as were demonstrated by typical apoptotic morphology, loss of mitochondrial outer membrane potential (reduction of DiOC6 uptake), increase of cells in subG1 fraction by propidium iodide (PI) staining, DNA fragmentation and caspase-3 activation. However, when we inhibit caspase activity by zVAD-fmk (zVAD), a pan-caspase inhibitor, two modes of cell death execution were observed. In K562, KT-1 and BV173 cells treated with INNO-406, zVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic, so-called caspase-independent necrosis-like cell death (CIND). While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases’ activity, the inhibition of the cell death by zVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of alter cell death pathway which is caspase-independent apoptosis (CIA) in MYL and Ba/F3/wt bcr-abl. The propensity towards CIND or CIA in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity, that is CIND with a high apoptosome activity potential while CIA with low. Freshly isolated leukemic cell samples from Bcr-Abl+ leukemia patients also had either low or high apoptosome activity potential. Moreover, cells undergoing CIND exhibited hallmarks of autophagy (i.e. the autophagosome formation, punctate formations of LC3 and the accumulation of LC3-II isoform), suggested the participation of autophagy in response to Bcr-Abl blockade. Inhibition of autophagy with chloroquine enhanced INNO-406-induced cell death, which indicates that the autophagic response of the tumor cells is protective. While, in vivo CML model, INNO-406 treatment increased apoptotic cells regardless of the caspase-3 activation, further implicating the involvement of caspase-independent cell death regulatory pathway in vivo in primary Bcr-Abl+ leukemic cells. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis depends on pulmonary metastases, which arise from multi-step progression of malignant tumors. We herein aimed to clarify the critical step of pulmonary metastasis using the syngeneic mouse spontaneous highly metastatic OS LM8 and parental Dunn cell lines, to identify new candidate molecules to suppress pulmonary metastasis. We first investigated the chronological detection of circulating tumor cells (CTCs) from mice with either cell line. LM8 CTCs appeared faster, at a higher rate and with a greater number compared to Dunn CTCs. Cultured cells from CTCs of LM8 showed higher proliferative ability than cells from the primary site in suspension culture, which mimicked the environment of the bloodstream for CTCs. The proliferative ability of LM8 cells was also higher than that of Dunn cells in 3D collagen culture with low stiffness (−150 Pa; close to conditions in the lung). We next focused on the extravasation step. LM8 showed higher migration ability compared to Dunn with transendothelial migration assay. We also found a disruption in endothelial barrier function throughout co-culture with LM8 using time-lapse imaging. In addition, LM8 secreted high levels of vascular endothelial growth factor (VEGF), while VEGF signal inhibition with a small molecule tyrosine kinase inhibitor (pazopanib) decreased disruption of the vascular barrier and transendothelial migration of LM8. Finally, daily oral administration of pazopanib reduced the rate and size of pulmonary metastasis in vivo. Collectively, these results show anti-VEGF therapy as a candidate for pulmonary metastasis of OS. Electronic supplementary material The online version of this article (doi:10.1007/s10585-012-9543-8) contains supplementary material, which is available to authorized users.