Yekaterina A. Miroshnikova

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

Tissue mechanics promote IDH1-dependent HIF1α-tenascin C feedback to regulate glioblastoma aggression.

Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status.

A 3D tension bioreactor platform to study the interplay between ECM stiffness and tumor phenotype.

Extracellular matrix (ECM) structure, composition, and stiffness have profound effects on tissue development and pathologies such as cardiovascular disease and cancer. Accordingly, a variety of synthetic hydrogel systems have been designed to study the impact of ECM composition, density, mechanics, and topography on cell and tissue phenotype. However, these synthetic systems fail to accurately recapitulate the biological properties and structure of the native tissue ECM. Natural three dimensional (3D) ECM hydrogels, such as collagen or hyaluronic acid, feature many of the chemical and physical properties of tissue, yet, these systems have limitations including the inability to independently control biophysical properties such as stiffness and pore size. Here, we present a 3D tension bioreactor system that permits precise mechanical tuning of collagen hydrogel stiffness, while maintaining consistent composition and pore size. We achieve this by mechanically loading collagen hydrogels covalently-conjugated to a polydimethylsiloxane (PDMS) membrane to induce hydrogel stiffening. We validated the biological application of this system with oncogenically transformed mammary epithelial cell organoids embedded in a 3D collagen I hydrogel, either uniformly stiffened or calibrated to create a gradient of ECM stiffening, to visually demonstrate the impact of ECM stiffening on transformation and tumor cell invasion. As such, this bioreactor presents the first tunable 3D natural hydrogel system that is capable of independently assessing the role of ECM stiffness on tissue phenotype.

STAT3 Blockade Inhibits Radiation-Induced Malignant Progression in Glioma

High grade gliomas (HGG) are classified into four subgroups based on transcriptional signatures and phenotypic characteristics. In particular, the proneural-to-mesenchymal transition (PMT) is associated with increased malignancy, poor prognosis, and disease recurrence, but the underlying causes of PMT are still unclear. In this study, we investigated whether radiotherapy promotes PMT using a genetically engineered mouse model of proneural HGG. We found that cranial ionizing radiation induced robust and durable PMT in tumors. Additionally, we isolated primary proneural HGG cells from mouse and human tumors and demonstrate that radiation induced a sustained cell-intrinsic mesenchymal transition associated with increased invasiveness and resistance to the alkylating agent temozolomide. Expectedly, irradiation-induced PMT was also associated with activation of the STAT3 transcription factor, and the combination of STAT3 blockade using JAK2 inhibitors with radiation abrogated the mesenchymal transition and extended survival of mice. Taken together, our data suggest that clinical JAK2 inhibitors should be tested in conjunction with radiation in patients with proneural HGG as a new strategy for blocking the emergence of therapy-resistant mesenchymal tumors at relapse.

Antisecretory Factor-mediated Inhibition of Cell Volume Dynamics Produces Anti-tumor Activity in Glioblastoma

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AFs ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777–90. ©2018 AACR.

Abstract PR05: Glycoprotein-mediated tissue mechanics regulate glioblastoma aggression

Glioblastoma multiforme (GBM) is a malignant glioma whose progression is associated with rampant extracellular matrix (ECM) remodeling. We recently found that GBM ECM stiffness predicts reduced survival in human patients. Instead of collagen fibrosis, which is common in many solid tumors, we showed that GBM stiffening involves increased production of extracellular glycoproteins, glycosaminoglycans, and sugar-binding proteins. Using bioinformatics, we revealed that genes of the glycocalyx (transmembrane glycoproteins and their interacting partners) are disproportionately upregulated in GBM relative to lower grade gliomas. Further, these genes are overexpressed within GBM in the mesenchymal (MES) relative to the proneural (PRO) subtype, the former of which is associated with treatment resistance and relapse. Using mouse models of human GBM, we showed that MES tumors are more lethal than PRO, and present with elevated ECM stiffness and mechanical signaling. To test our hypothesis that mechanical signaling can drive the MES phenotype, we engineered PRO GBM cells with constitutively-elevated integrin signaling. Compared to control PRO cells, these undergo a robust MES-like transition, upregulate bulky glycoprotein expression, and result in stiffer and more lethal tumors. This phenotype was reversed by the inhibition of focal adhesion kinase in MES cells. To test whether an enhanced glycocalyx can directly elevate mechanical signaling, we decorated GBM cells with synthetic glycoprotein polymers. Indeed, this resulted in enhanced integrin-focal adhesion signaling and more aggressive tumor progression. The invasive properties and therapy resistance observed in mesenchymal tumor cells are often associated with elevated stem cell-like features. To investigate a link between the glycocalyx, tissue mechanics, and the mesenchymal-stem cell phenotype, we interfered with components of the gylcocalyx or mechanical signaling machinery and found a reduction in stem cell genes and surface proteins, as well as increased sensitivity to chemotherapy. These data support a model in which glycoprotein-mediated tissue stiffening drives GBM aggression through promotion of a mesenchymal phenotype. This abstract is also being presented as Poster A39. Citation Format: J. Matthew Barnes, Elliot C. Woods, Russell O. Bainer, Yekaterina A. Miroshnikova, Kan Lu, Gabriele Bergers, Carolyn Bertozzi, Valerie M. Weaver. Glycoprotein-mediated tissue mechanics regulate glioblastoma aggression. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR05.

Abstract PR10: A glycoprotein-mediated mechanical switch promotes glioma aggression

Glioblastoma multiforme (GBM) is a malignant brain tumor whose progression is associated with rampant extracellular matrix (ECM) remodeling. We recently found that ECM stiffness correlates with poor survival in human GBM specimens. Glycoproteins are the major constituent of normal brain ECM and many are overexpressed in brain tumors, yet the interplay between glycoproteins and mechanical signaling in GBM pathogenesis remains poorly understood. Here, we show that bulky glycoproteins and sugar-binding proteins are broadly upregulated in GBM relative to lower grade gliomas. Further, these genes are overexpressed in the mesenchymal (Mes) relative to the proneural (Pro) GBM subclass, the former of which is associated with treatment resistance and relapse. We took a specific interest in the hyaluronic acid (HA)-producing enzyme, HAS2, and the galactoside-binding lectin galectin-1 (Gal1) due to their ability to modulate tissue structure and rheology. Using mouse models of human GBM we showed that Mes tumors are enriched in HA and fibronectin, coincident with elevated ECM stiffness and mechanical signaling. These data suggest the possibility that aberrant glycoprotein expression drives GBM aggression through enhanced mechanical signaling resulting from tissue stiffening. Consistent with this hypothesis, by elevating mechanical signaling in Pro GBMs we induce a robust Mes-like transition and we see the opposite when reducing Gal1 expression or HA content in Mes tumors. Our data provides evidence of a feed-forward mechanism whereby mechanical signaling drives Gal1 and HA production which reinforce ECM stiffness, thus sustaining pro-tumorigenic mechanical signaling. This abstract is also presented as Poster A21. Citation Format: J Matthew Barnes, Yekaterina A. Miroshnikova, Jason C. Tung, Russel O. Bainer, Valerie M. Weaver. A glycoprotein-mediated mechanical switch promotes glioma aggression. [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 PR10.

Abstract LB-143: Interplay between tissue tension and inflammation in pancreatic tumor progression

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies due largely to an inability to detect these tumors early and thereafter a lack of effective therapies for late stage cancers (Tempero, 2003). PDAC is characterized by a strong desmoplastic response including significant extracellular matrix (ECM) remodeling that severely compromises treatment and surgical resection (Farrow, 2008). Nevertheless, the molecular mechanisms linking tissue transformation to fibrosis and tumor aggression remain unclear. Tissue fibrosis increases tissue tension and we showed that ECM stiffness and epithelial cell tension drive squamous carcinoma and mammary transformation (Levental, 2009; Samuel, 2011). We therefore asked whether pancreatic fibrosis promotes PDAC progression and aggression by increasing ECM stiffness and tissue tension and how. Inflammation is casually-associated with fibrosis and targeted deletion of the TGFβ receptor 2 (TGFβR2KO) when combined with expression of KRas up regulates CXC chemokine expression to promote PDAC aggression (Ijichi, 2011). Here we report that PDAC progression is accompanied by significant fibrosis and inflammation that correlated strongly with collagen deposition and LOX-dependent cross-linking and ECM stiffening. PDAC progression associated with elevated CXC chemokine levels as well as high epithelial STAT3 activity and elevated mechanosignaling in the epithelium including activated myosin, FAK and YAP and enhanced PDAC contractility. Intriguingly, we showed that PDAC contractility is functionally linked through a reciprocal Cxcr2 receptor - GPCR- JAK-STAT3 - ROCK - FAK signaling circuit that contributes to PDAC aggression and tissue inflammation. Consistently, the pancreas of mice expressing a targeted β1 integrin V737lox/lox clustering mutant, which drives integrin-dependent mechanosignaling in their epithelium showed profound fibrosis, high pSTAT3 and chronic inflammation which progressed to pancreatitis by as early as 3 months of age when combined with the KRas oncogene. Ongoing studies are currently exploring the relevance of Stat3 on pancreatic fibrosis and tumor aggression. Nevertheless, our findings identify a vicious positive feedback mechanism mediated through tissue tension that may account for the strong association between fibrosis, tumor aggression and inflammation in PDAC. (This work was supported by the Tumor Microenvironment Network (TMEN) grant NIH/NCI 1U01 CA151925-01, the Pancreatic Cancer Action Network-AACR Innovative Grant 30-60-25-WEAV, and NIH/NCI R01 CA138818-01A1 to VMW). Citation Format: Hanane Laklai, Yekaterina Miroshnikova, Jon lakins, Novitskiy Sergey, Agnes Gorska, Lidiya Korets, Raghu Kalluri, Harold Moses, Valerie Weaver. Interplay between tissue tension and inflammation in pancreatic tumor progression. [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 LB-143. doi:10.1158/1538-7445.AM2014-LB-143

Abstract A47: Reciprocal dialogue between cellular and extracellular matrix tension and tissue inflammation in pancreatic tumor progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound fibrotic response that contributes to tumor aggression and treatment resistance (Farrow et al., Journal of Surgical Research, 2008). Yet, the molecular mechanisms linking tissue transformation to fibrosis and tumor aggression remain unclear. Tissue fibrosis promotes tissue stiffening and we showed that tissue tension drives transformation and cancer progression, thus we studied the relationship between PDAC progression, fibrosis, and extracellular matrix (ECM) stiffening using KRas transgenic mice (Levental et al., Cell, 2009; Samuel et al., Cancer Cell, 2011). Because inflammation has been casually associated with fibrosis, we combined KRas-induced carcinogenesis with targeted deletion of the TGFβ receptor 2 (TGFβR2KO) which potentiates PDAC aggression through upregulation of Cxc chemokine expression and elevated Stat3 signaling (Ijichi et al., Journal of Clinical Investigation, 2011). Here we report that PDAC progression is accompanied by collagen deposition, reorganization, and LOX-dependent cross-linking that stiffens the ECM and activates focal adhesion kinase (FAK) and that is substantially potentiated through loss of TGFβ receptor 2 signaling. We also noted that the pancreatic epithelium in the TGFβ R2KO mice had higher levels of activated myosin and exerted greater traction forces that enhanced collagen remodeling and contraction. Consistent with a functional link between chemokine signaling and ROCK-dependent cell contractility we showed that the contraction phenotype of TGFβ R2KO pancreatic epithelial cancer cells depends upon Cxcr2 receptor signaling and JAK-ROCK activity (Sanz-Moreno et al., Cancer Cell, 2011). Intriguingly, we also found that Cxc chemokine expression is greatly potentiated by ECM stiffness which feeds forward to enhance Stat3 activation. These findings identify a vicious positive feedback mechanism mediated through ECM remodeling and stiffening that may account for the association between fibrosis, tumor aggression and inflammation in PDACs. This work was supported by the Tumor Microenvironment Network (TMEN) grant NIH/NCI 1U01 CA151925-01 and the Physical Sciences Oncology Center (PSOC) NIH/NCI U54CA143836-01. Citation Format: Hanane Laklai, Raghu Kalluri, Harold Moses, Valerie Weaver, Hideaki Ijichi, Jonathan Lakins, Yekaterina Miroshnikova, Irene Acerbi, Jose Lopez, Elena Kassianidou, Agnieszka Gorska, Sergey Novitskiy. Reciprocal dialogue between cellular and extracellular matrix tension and tissue inflammation in pancreatic tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A47.

Abstract LB-96: Extrinsic and intrinsic force and GBM aggression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Despite concerted effort the mean survival time for patients with aggressive GBM remains unchanged with a mean of 14 months. Indeed, high grade GBMs are notoriously resistant to therapy and typically disseminate throughout the brain tissue, severely compromising patient treatment. In this respect, the most lethal GBMs arise within the SVZ region of the brain which is rich in stem cells. This has lead to speculation that GBM aggression is related to their stem-like origin. Consistently, using AFM indentation we determined that analogous to neural stem cells (NSCs), high grade GBMs are very soft and express high levels of the Notch regulated gene HES-1. We also found that high grade GBMs and NSCs both express elevated levels of the stemness-promoting repressor NCoR2. We further determined that NCoR2 expression correlates with poor GBM patient prognosis, which is an observation that accords with our recent finding that NCoR2 enhances breast tumor survival in response to radiation, chemotherapy and immune activators in vitro and in vivo. These findings are consistent with a stem-like origin for GBMs and identify NCoR2 as a putative unique survival mechanism that could explain the treatment resistance of this disease. Interestingly, AFM measurements of freshly excised murine brain tumor slices showed that GBMs arising within the SVZ region are also quite stiff. Preliminary findings also showed that isolated high grade GBM cells but not low grade oligodendroglioma cells have a greatly enhanced mechano-responsiveness e.g. they spread more on soft gels and possess a vastly altered glycocalyx; traits that predict elevated contractility and integrin signaling. Given MRI data indicating very aggressive GBMs in the SVZ region are highly vascularized and experience elevated compression the findings support the idea that tumors that arise within this highly mechanically-challenged SVZ microenvironment have a unique mechano-behavior that could facilitate their growth, survival and dissemination. Indeed, the high compliance, contractility and mechanical resistance of GBMs would facilitate their growth, survival and expansion within compressed brain tissue and would foster their ability to navigate and disseminate into the dense GBM-associated ECM. Because reducing tumor force can revert the malignant phenotype of cancerous tissue and will reduce tumor cell invasion, growth, and survival and inhibit tumor progression and decrease tumor incidence we predict that the altered force microenvironment and intrinsic mechanobehavior of GBMs contribute to their aggression. Consequently, studies are now underway to clarify the interplay between cell and tissue force and GBM behavior and to determine the relevance of NCoR2 to GBM treatment resistance. (Supp by: NCI grants U54CA143836–01 and NIH/NCI R01 CA138818–01A1 to V.M.W. and a National Science Foundation (GRFP) Fellowship to Y.A.M.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-96. doi:10.1158/1538-7445.AM2011-LB-96