Glioblastoma cellular cross-talk converges on NF-κB to attenuate EGFR inhibitor sensitivity

Funding Information: We thank Dr. David James, Dr. Frederick Lang, Dr. Cameron Brennan, and Dr. Harley Kornblum for GBM-PDX neurospheres. We thank Dr. Karen Arden for continuous support and critical evaluation of the results. We thank Dr. Robert Davis, Dr. German Gomez, Dr. Tiffany Taylor, Dr. Rachel Reed, Dr. Melissa Mcalonis, and Dr. Sora Lee for technical support. In memory of Rosa Lupo. This work was supported by the Defeat GBM Research Collaborative, a subsidiary of the National Brain Tumor Society (F.B.F. and P.S.M.), R01-NS080939 (F.B.F.), the James S. McDonnell Foundation (F.B.F.), the National Cancer Institute (2T32CA009523-29A1) (A.H.T), and 1RO1NS097649-01 (C.C.C.). C.Z. was partially supported by an American-Italian Cancer Foundation post-doctoral research fellowship. F.L. received a Gao Feng Gao Yuan Scholarship Award. T.C.G., A.K.S., P.S.M., W.K.C., and F.B.F. receive salary and additional support from the Ludwig Institute for Cancer Research. Publisher Copyright: © 2017 Zanca et al.In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.publishersversionPeer reviewe

Quantifying Cellular Adhesion Strength of Tumor Cells as a Metric for Migratory and Metastatic Potential

The ability of tumor cells to reestablish a niche and cause recurrence of the tumor, either within the primary tissue that the tumor initially formed or to a secondary site within the body, enhances tumor malignancy and causes patient morbidity and mortality. The propensity of tumor cells to migrate away from their primary site poses several challenges. First is the high variability in driver mutations between patients, as well as the variability in patient background genetics,both of which can induce variability in tumor cell migratory and metastatic propensity. Second, is a lack of universal markers, either genetic or molecular, which can accurately predict tumor cell migratory and metastatic potential. Together these challenges prevent the stratification of different tumors and prevents the implementation of patient-specific interventions tailored to prevent tumor cell migration and metastasis.An emerging field which is providing insight into the tumor metastatic process is biophysical analysis. However, the field is currently focused on analyzing the presence of circulating tumor cell through turbulent flow microfluidics as an indicator for metastasis, not predicting tumor metastatic potential from the primary site. Therefore, there is a need for a rapid and quantitative biophysical metric which can predict the migratory and metastatic potential of those primary tumors. This dissertation addresses the need to predict tumor migratory and metastatic potential by gaining an understanding of how those characteristics are linked to cell adhesion in glioblastoma and mammary tumors, respectively.In order to determine the relationship between adhesion strength and cell migratory and metastatic potential first we characterized the adhesion strength of metastatic and non-metastatic mammary cancer cells. In order to evaluate cellular adhesion strength as well as understand the effects of the tumor microenvironment on cell biophysical characteristics, we built a spinning disk shear device. This device gave us the capability of interrogating cellular adhesion characteristics in a quantitative and high throughput manner, as well as being able to modulate extracellular divalent cation conditions Mg2+ and Ca2+ to mimic those found in patients. We found that in the absence of divalent cations those metastatic cells showed an overall decrease in the cellular adhesion strength as well as a broader range of adhesion characteristicsthan those non-metastatic cell lines. Comparison of those metastatic cells to their non-metastatic counterparts demonstrated a decrease in the assembly state of focal adhesions in both number and size. Similarly, the exposure of those non-metastatic cells to cyclic RGD peptides also induced a decrease in focal adhesion assembly state as well as decreasing cellular adhesion strength and increasing cell migratory phenotype. Together, these data suggest that there is a correlation between decreased cellular adhesion strength, an increase in metastatic potential, and that this correlation is due to altered assembly state of focal adhesion structures.Next, I wanted to understand if the correlation between decreased cellular adhesion strength and increased cell migratory phenotype extended beyond mammary tumors to glioblastoma (GBM). In order to investigate the effects of common GBM mutations on cellular adhesion strength, isogenic murine astrocytes were exposed to fluidic shear stress via spinning disk assay. Specifically, astrocytes with combinations of CDKN2A/B deletion (occurring in 61% of patients), Pten deletion (occurring in 41% of patients), or alteration of epidermal growth factor receptor (EGFR) (occurring in 57% of patients) were analyzed. This analysis showed that unlike mammary tumors, decreased astrocyte adhesion strength was dependent on the presence of divalent cations. Furthermore, I found that the decrease in adhesion strength was limited to those cells expressing EGFRvIII independent of other mutations, and correlated with increased migratory phenotype. Further analysis found that this change in EGFRvIII expressing cells biophysical phenotype is a result of a signaling-dependent decrease in integrin expression which results in alteration of focal adhesion assembly state. In order to investigate what EGFRvIII dependent signaling cascades modulate decreased adhesion strength, I utilized small-molecule inhibitors to target multiple pathway nodes such as: the EGFR receptor itself, MEK, SRC, and Stat3in a systematic manner. I found that those cells treated with MEK inhibitor or EGFR inhibitor showed an increase in EGFRvIII cell adhesion strength, similar to non-EGFRvIII expressing cells. Lastly, it has been well documented that cell-cell communication allows those EGFRvIII cells to affect behavior of non-EGFRvIII cells within that tumor. In order to understand if EGFRvIII cells are capable of altering cell adhesion strength of other cells, I educated wtEGFR with EGFRvIII conditioned media prior to analyzing cellular adhesion strength. I found that those wtEGFR expressing cells altered their adhesion strength after education with vIII CM, and that this decrease in adhesion strength was dependent on the presence of the soluble factor TNF-α.Throughout this dissertation I will demonstrate the value of utilizing fluidic shear as a methodology for analyzing cellular adhesion strength and its application for predicting tumor migratory and metastatic potential. Furthermore, I will also demonstrate how fluidic shear can be used to understand the genetic and molecular mechanisms that contribute to and enhance tumor cell malignancy

Metastatic State of Cancer Cells May Be Indicated by Adhesion Strength.

Cancer cells within a tumor are heterogeneous and only a small fraction are able to form secondary tumors. Universal biological markers that clearly identify potentially metastatic cells are limited, which complicates isolation and further study. However, using physical rather than biological characteristics, we have identified Mg2+- and Ca2+-mediated differences in adhesion strength between metastatic and nonmetastatic mammary epithelial cell lines, which occur over concentration ranges similar to those found in tumor stroma. Metastatic cells exhibit remarkable heterogeneity in their adhesion strength under stromal-like conditions, unlike their nonmetastatic counterparts, which exhibit Mg2+- and Ca2+-insensitive adhesion. This heterogeneity is the result of increased sensitivity to Mg2+- and Ca2+-mediated focal adhesion disassembly in metastatic cells, rather than changes in integrin expression or focal adhesion phosphorylation. Strongly adherent metastatic cells exhibit less migratory behavior, similar to nonmetastatic cell lines but contrary to the unselected metastatic cell population. Adhesion strength heterogeneity was observed across multiple cancer cell lines as well as isogenically, suggesting that adhesion strength may serve as a general marker of metastatic cells

Adhesion strength and contractility enable metastatic cells to become adurotactic.

Significant changes in cell stiffness, contractility, and adhesion, i.e., mechanotype, are observed during a variety of biological processes. Whether cell mechanics merely change as a side effect of or driver for biological processes is still unclear. Here, we sort genotypically similar metastatic cancer cells into strongly adherent (SA) versus weakly adherent (WA) phenotypes to study how contractility and adhesion differences alter the ability of cells to sense and respond to gradients in material stiffness. We observe that SA cells migrate up a stiffness gradient, or durotax, while WA cells largely ignore the gradient, i.e., adurotax. Biophysical modeling and experimental validation suggest that differences in cell migration and durotaxis between weakly and strongly adherent cells are driven by differences in intra-cellular actomyosin activity. These results provide a direct relationship between cell phenotype and durotaxis and suggest how, unlike other senescent cells, metastatic cancer cells navigate against stiffness gradients

Adhesion strength and contractility enable metastatic cells to become adurotactic.

Significant changes in cell stiffness, contractility, and adhesion, i.e., mechanotype, are observed during a variety of biological processes. Whether cell mechanics merely change as a side effect of or driver for biological processes is still unclear. Here, we sort genotypically similar metastatic cancer cells into strongly adherent (SA) versus weakly adherent (WA) phenotypes to study how contractility and adhesion differences alter the ability of cells to sense and respond to gradients in material stiffness. We observe that SA cells migrate up a stiffness gradient, or durotax, while WA cells largely ignore the gradient, i.e., adurotax. Biophysical modeling and experimental validation suggest that differences in cell migration and durotaxis between weakly and strongly adherent cells are driven by differences in intra-cellular actomyosin activity. These results provide a direct relationship between cell phenotype and durotaxis and suggest how, unlike other senescent cells, metastatic cancer cells navigate against stiffness gradients

EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration.

A lack of biological markers has limited our ability to identify the invasive cells responsible for glioblastoma multiforme (GBM). To become migratory and invasive, cells must downregulate matrix adhesions, which could be a physical marker of invasive potential. We engineered murine astrocytes with common GBM mutations, e.g. Ink4a (Ink) or PTEN deletion and expressing a constitutively active EGF receptor truncation (EGFRvIII), to elucidate their effect on adhesion. While loss of Ink or PTEN did not affect adhesion, counterparts expressing EGFRvIII were significantly less adhesive. EGFRvIII reduced focal adhesion size and number, and these cells - with more labile adhesions - displayed enhanced migration. Regulation appears to depend not on physical receptor association to integrins but, rather, on the activity of the receptor kinase, resulting in transcriptional integrin repression. Interestingly, EGFRvIII intrinsic signals can be propagated by cytokine crosstalk to cells expressing wild-type EGFR, resulting in reduced adhesion and enhanced migration. These data identify potential intrinsic and extrinsic mechanisms that gliomas use to invade surrounding parenchyma

EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration.

A lack of biological markers has limited our ability to identify the invasive cells responsible for glioblastoma multiforme (GBM). To become migratory and invasive, cells must downregulate matrix adhesions, which could be a physical marker of invasive potential. We engineered murine astrocytes with common GBM mutations, e.g. Ink4a (Ink) or PTEN deletion and expressing a constitutively active EGF receptor truncation (EGFRvIII), to elucidate their effect on adhesion. While loss of Ink or PTEN did not affect adhesion, counterparts expressing EGFRvIII were significantly less adhesive. EGFRvIII reduced focal adhesion size and number, and these cells - with more labile adhesions - displayed enhanced migration. Regulation appears to depend not on physical receptor association to integrins but, rather, on the activity of the receptor kinase, resulting in transcriptional integrin repression. Interestingly, EGFRvIII intrinsic signals can be propagated by cytokine crosstalk to cells expressing wild-type EGFR, resulting in reduced adhesion and enhanced migration. These data identify potential intrinsic and extrinsic mechanisms that gliomas use to invade surrounding parenchyma

EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration.

A lack of biological markers has limited our ability to identify the invasive cells responsible for Glioblastoma multiforme. To become migratory and invasive, cells must down regulate matrix adhesions, which could be a physical marker of invasive potential. Murine astrocytes were engineered with common GBM mutations, e.g. Ink4a (Ink) or PTEN deletion and expressing a constitutively active EGF receptor truncation (i.e. EGFRvIII), to elucidate their effect on adhesion. While loss of Ink or PTEN did not affect adhesion, counterparts expressing EGFRvIII were significantly less adhesive. EGFRvIII reduced focal adhesion size and number, and these cells with more labile adhesions displayed enhanced migration. Regulation appears dependent not on physical receptor association to integrins but rather on the receptor's kinase activity resulting in transcriptional integrin repression. Interestingly, EGFRvIII intrinsic signals can be propagated by cytokine crosstalk to wildtype EGFR cells, resulting in reduced adhesion and enhanced migration. These data identify potential intrinsic and extrinsic mechanisms that gliomas use to invade surrounding parenchyma