Pro-invasive Signaling Research Articles

Emerging Roles of Small Extracellular Vesicles in Gastrointestinal Cancer Research and Therapy

Discovered in the late eighties, sEVs are small extracellular nanovesicles (30–150 nm diameter) that gained increasing attention due to their profound roles in cancer, immunology, and therapeutic approaches. They were initially described as cellular waste bins; however, in recent years, sEVs have become known as important mediators of intercellular communication. They are secreted from cells in substantial amounts and exert their influence on recipient cells by signaling through cell surface receptors or transferring cargos, such as proteins, RNAs, miRNAs, or lipids. A key role of sEVs in cancer is immune modulation, as well as pro-invasive signaling and formation of pre-metastatic niches. sEVs are ideal biomarker platforms, and can be engineered as drug carriers or anti-cancer vaccines. Thus, sEVs further provide novel avenues for cancer diagnosis and treatment. This review will focus on the role of sEVs in GI-oncology and delineate their functions in cancer progression, diagnosis, and therapeutic use.

Loss of YY1, a Regulator of Metabolism in Melanoma, Drives Melanoma Cell Invasiveness and Metastasis Formation.

Deregulation of cellular metabolism through metabolic rewiring and translational reprogramming are considered hallmark traits of tumor development and malignant progression. The transcription factor YY1 is a master regulator of metabolism that we have previously shown to orchestrate a metabolic program required for melanoma formation. In this study, we demonstrate that YY1, while being essential for primary melanoma formation, suppresses metastatic spreading. Its downregulation or loss resulted in the induction of an invasiveness gene program and sensitized melanoma cells for pro-invasive signaling molecules, such as TGF-β. In addition, NGFR, a key effector in melanoma invasion and phenotype switching, was among the most upregulated genes after YY1 knockdown. High levels of NGFR were also associated with other metabolic stress inducers, further indicating that YY1 knockdown mimics a metabolic stress program associated with an increased invasion potential in melanoma. Accordingly, while counteracting tumor growth, loss of YY1 strongly promoted melanoma cell invasiveness in vitro and metastasis formation in melanoma mouse models in vivo. Thus, our findings show that the metabolic regulator YY1 controls phenotype switching in melanoma.

Abstract 2937: Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth

Abstract The goal of this study is to define the role of NEDD9 in formation of progression of non-small cell lung cancer (NSCLC). NSCLC has a low survival rate, with metastasis contributing to the vast majority of deaths. NEDD9 expression has been shown to be repressed by LKB1, and upregulated in LKB1-mutated NSCLC; in these tumors, elevated NEDD9 promotes metastasis, serving as a scaffolding intermediate in the integrin and receptor tyrosine kinase signaling cascades, promoting pro-invasive signaling. In this study, we for the first time assessed the consequences for cancer formation after introduction of a constitutive null allele of Nedd9 (KPN mice) in the Krastm4Tyj/J/Trp53tm1Brn/J (KP mice) Adeno-cre inducible murine model of NSCLC, in the context of intact Lkb1. Unexpectedly, KPN mice presented with significantly accelerated NSCLC tumorigenesis and enhanced tumor invasion. Orthotopic re-implantation of KP and KPN tumor into syngeneic mice with wt and null Nedd9 genotypes demonstrated that the effect of Nedd9 loss on promoting tumor growth is cell-autonomous. Proteomic analysis of KPN versus KP tumors revealed that more aggressive phenotypes occurred in spite of depressed activity of the pro-proliferative, pro-invasive Nedd9 partner proteins Src and Fak. Treatment of mice with the Src inhibitor dasatinib significantly reduced, but did not eliminate, tumor growth in KP and KPN mice. As an alternative hypothesis, we considered that Ras-dependent NSCLC tumors require active autophagy, a process dependent on activation of the kinases Lkb1 and Ampk; given Lkb1 represses Nedd9, we investigated whether Nedd9 reciprocally regulates Lkb1. We found that KPN tumors specifically and significantly upregulated active Lkb1 based on a mechanism of post-transcriptional activation, causing increased expression of the Lkb1 substrate Ampk and downstream effectors of autophagy, including elevated appearance of LC3-positive autophagosomes in KPN NSCLC tumor tissue. NEDD9 depletion in human NSCLC cell lines similarly elevated LKB1 activation. In vivo treatment of KPN and KP mice with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors, emphasizing the importance of autophagy for the growth differential. In sum, these data for the first time identify and define a previously unknown role for the pro-metastatic protein NEDD9 in control of autophagy, based on a role as a negative regulator of the Lkb1-Ampk signaling axis in NSCLC. Citation Format: Alexander Y. Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Anna Kiseleva, Douglas Flieder, Ilya Serebriiskii, Harvey Hensley, Erica Golemis. Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2937.

MODL-14. SMALL MOLECULE TARGETING OF ONCOGENIC FGF2-FGFR SIGNALING IN BRAIN TUMORS

FGF2, the ligand of FGF receptors (FGFRs), is expressed in the developing and adult brain. FGF2-FGFR1 signaling causes the induction and maintenance of cancer stem cells through ERK-dependent up-regulation of ZEB1 and Olig2 in glioblastoma. In SHH medulloblastoma, Olig2 triggers tumor initiation from GCPs, maintains quiescent stem-like cells during the disease and contributes to tumor outgrowth at recurrence. We found that FGF2-FGFR signaling causes increased growth and tissue invasion through the FGFR adaptor protein FRS2 in SHH and group-3 medulloblastoma 1. Thus, targeting of FGFR-FRS2 signaling could abrogate brain tumor growth and spread by repressing tumor-promoting functions that are induced by microenvironmental FGF2. Using virtual screening combined with functional validation, we identified protein-protein interaction inhibitors (F2i) that bind FRS2 and abrogate FGFR signaling to the MAP-ERK pathway. Consistent with the requirement of FRS2 for pro-invasive signaling downstream of FGFR1 in medulloblastoma, F2i also efficiently block FGF2-induced migration and invasion in medulloblastoma-derived cells. Selected F2i display excellent binding kinetics with a similar Kd as the natural ligand domain of FGFR and cause steric alterations in the targeted protein domain. On-target activity was confirmed by thermal proteome profiling. Neither in silico screening nor empirical testing revealed significant off-target activity of the compounds. No toxicity of F2i was observed in cell-based models with confirmed functional activity on invasion and MAPK activation. Thus, we identified novel, low molecular weight pharmacological protein-protein interaction inhibitors with an excellent potential to specifically block FGFR functions relevant for brain tumor progression. 1. Santhana Kumar et al., CellReports23, 3798–3812.e8 (2018).

SNAIL Transctiption factor in prostate cancer cells promotes neurite outgrowth

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.

Epidermal Growth Factor (EGF) Augments the Invasive Potential of Human Glioblastoma Multiforme Cells via the Activation of Collaborative EGFR/ROS-Dependent Signaling.

Abnormal secretion of epidermal growth factor (EGF) by non-neuronal cells (e.g., glioma-associated microglia) establishes a feedback loop between glioblastoma multiforme (GBM) invasion and a functional disruption of brain tissue. Considering the postulated significance of this vicious circle for GBM progression, we scrutinized mechanisms of EGF-dependent pro-invasive signaling in terms of its interrelations with energy metabolism and reactive oxygen species (ROS) production. The effects of EGF on the invasiveness of human glioblastoma T98G cells were estimated using time-lapse video microscopy, immunocytochemistry, cell cycle assay, immunoblot analyses, and Transwell® assay. These techniques were followed by quantification of the effect of EGFR (Epidermal Growth Factor Receptor) and ROS inhibitors on the EGF-induced T98G invasiveness and intracellular ROS, ATP, and lactate levels and mitochondrial metabolism. The EGF remarkably augmented the proliferation and motility of the T98G cells. Responses of these cells were accompanied by cellular rear–front polarization, translocation of vinculin to the leading lamellae, and increased promptness of penetration of micropore barriers. Erlotinib (the EGFR inhibitor) significantly attenuated the EGF-induced T98G invasiveness and metabolic reprogramming of the T98G cells, otherwise illustrated by the increased mitochondrial activity, glycolysis, and ROS production in the EGF-treated cells. In turn, ROS inhibition by N-acetyl-L-cysteine (NAC) had no effect on T98G morphology, but considerably attenuated EGF-induced cell motility. Our data confirmed the EGFR/ROS-dependent pro-neoplastic and pro-invasive activity of EGF in human GBM. These EGF effects may depend on metabolic reprogramming of GBM cells and are executed by alternative ROS-dependent/-independent pathways. The EGF may thus preserve bioenergetic homeostasis of GBM cells in hypoxic regions of brain tissue.

Blocking pro-invasive signaling and inflammatory activation in triple-negative breast cancer with nucleic-acid scavengers (NASs).

e13096 Background: Breast cancers remain the most lethal malignancies amongst women worldwide and the second leading cause of cancer-related mortalities in the US. Subtype heterogeneity and aggressive invasive potential are believed to be the major contributors of these outcomes. Triple-negative breast cancer (TNBC) are notoriously aggressive, difficult-to-treat, and metastatic. Inflammation-driven tumorigenesis has been shown to correlate with cell-free DNA (cfDNA) and other damage-associated molecular patterns (DAMPs) in cancer patient sera. We showed that nucleic-acid scavengers (NAS) can block pro-inflammatory signals elicited by DAMP-activation of innate immune sensors (e.g. toll-like receptors). Treatment with the NAS PAMAM-G3 drastically reduced liver metastatic burden in an immunocompetent murine model of pancreatic cancer. Methods: TNBC cells lines were treated with a cocktail of standard-of-care chemotherapeutic agents and the conditioned media (CM) from these cells served as an in vitro DAMP source. Downstream function of TLR activation was tested via a HEK293-TLR reporter cell line measuring absorbance at 655nm. The in vitro invasive phenotype was tested and quantified using a Transwell-Matrigel invasion assay. Cytokine secretion was measured using a BioLegend cytokine array. Results: TNBC CM greatly increased TNBC cell invasion in vitro and that treatment with the NAS PAMAM-G3 significantly inhibits this effect. Treatment of human monocytes (THP-1) with TNBC CM elicited a strong pro-inflammatory response with elevated levels of IL-8, IL-6, CCL2, and IL-1β. Other biologically immune responders including human PBMCs will be tested to determine the potential impact on the tumor immune microenvironment during tumorigenesis and treatment. Conclusions: To elucidate the mechanism by which this NAS works in these tumor settings, our lab has developed several PAMAM-G3 derivatives, including biotin, IR-, and near-IR fluorophore labeled molecules. These molecules will allow us to capture and characterize DAMPs and do in vivo live imaging experiments to gain insight into NAS PK/PD properties. This insight into NAS capabilities will enhance our understanding of metastatic progression and its interplay with the immune system. Moreover, these principles will aid in the development of novel of anti-metastatic therapies to improve TNBC patient outcomes.

Adrenergic receptors signal to β‐arrestins to control the membrane trafficking of Monocarboxylate transporter 1 (MCT1) in glioblastoma

Monocarboxylate transporters (MCTs) are a family of 12‐pass transmembrane domain proteins that mediate the rapid symport of protons and monocarboxylates, including lactate, across the plasma membrane. MCT‐1, the primary MCT in glioblastoma cells, helps regulate mammalian metabolism and is thought to act as a scaffold for and facilitate the activation of pro‐migratory and pro‐invasive signaling pathways. Uncovering the cellular mechanisms governing MCT‐1 trafficking is crucial for the development of therapeutic treatments for MCT‐1 as an anticancer target. Here, we describe the role of signaling by β2‐adrenergic (β2AR) and α2‐adrenergic (α2AR) receptors in mediating MCT‐1 internalization into caveolae and sorting endosomes. Through MCT‐1 and Caveolin‐1 colocalization analysis, we show an increase in MCT‐1 internalization upon β2AR agonist stimulation and even greater increase in internalization upon simultaneous β2AR and α2AR agonist stimulation, which suggest that both receptors mediate this response in a synergistic manner. Furthermore, we demonstrate that siRNA depletion of endogenous β‐arrestin inhibits MCT‐1 internalization in cells when stimulated with a β2AR agonist, indicating that β2AR likely signals through β‐arrestin to mediate MCT‐1 internalization. We are currently investigating whether these results are recapitulated in single‐cell derived β‐arrestin knockout clones. Our current research also focuses on identifying which β‐arrestin‐dependent signaling pathways promote synergistic MCT‐1 internalization in response to adrenergic receptor activation.

Abstract A90: Blocking proinvasive signaling and inflammatory activation in triple-negative breast cancer with nucleic-acid scavengers (NAS)

Abstract Breast cancers (BC) remain the most lethal malignancies among women worldwide and the second leading cause of cancer-related mortalities in the US. Subtype heterogeneity and aggressive invasive potential are believed to be the major contributors of these outcomes. BC lacking canonical histologic receptors (i.e., ER/PR/HER2), called triple negative (TNBC), are notoriously aggressive, difficult to treat, and metastatic. It has been observed that the degree of inflammation-driven tumorigenesis tends to correlate with increased levels of cell free DNA (cfDNA) and other damage-associated molecular patterns (DAMPs) in cancer patient sera. Our lab has previously shown that nucleic-acid scavengers (NAS) can block proinflammatory and proinvasive/metastatic signals elicited by nucleic acid-containing DAMP activation of innate immune sensors such as of the Toll-like receptors (TLRs). Recently, we showed that treatment with the nucleic acid-binding polymer, PAMAM-G3, resulted in a drastic reduction in metastatic tumor burden to the liver in an immunocompetent murine model of pancreatic cancer (Naqvi I, Gunaratne R, et al., Mol Ther 2018;26). Ongoing work has shown that chemotherapy-derived TNBC-conditioned media (CM) and TNBC patient sera greatly increase TNBC cell invasion in vitro and that treatment with the NAS PAMAM-G3 significantly inhibits this effect. Treatment of the human monocyte cell line, THP-1, with TNBC CM elicited a very strong proinflammatory response comparable to known innate immune stimulants such as poly(I:C), LPS, and CpG, with elevated levels of IL-8, IL-6, MCP-1 (CCL2), IL-18, and IL-1β. Other biologically relevant immune responders such as isolated human PBMCs and whole blood will be tested to determine the potential impact on the tumor immune microenvironment during tumorigenesis and treatment. To elucidate the mechanism by which this NAS works in these tumor settings, our lab has developed several PAMAM-G3 derivatives, including biotin, IR-, and near-IR fluorophore-labeled molecules. These molecules will allow us to conduct DAMP capture and characterization experiments, as well as perform in vitro and in vivo live imaging experiments to gain insight into NAS PK/PD properties. Mechanistic insight into NAS antimetastatic and anti-inflammatory capabilities will enhance our understanding of metastatic progression and its interplay with the immune system. Moreover, these principles will aid in the development of novel of antimetastatic therapies to improve TNBC patient outcomes. Citation Format: Elias O.U. Eteshola, Karenia Landa, E. Shelley Hwang, Angelo Moreno, Smita K. Nair, Bruce A. Sullenger. Blocking proinvasive signaling and inflammatory activation in triple-negative breast cancer with nucleic-acid scavengers (NAS) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A90.

Abstract 4515: Utilizing nucleic-acid scavengers (NASs) to inhibit proinflammatory and proinvasive signaling in triple-negative breast cancer

Abstract Breast cancers (BC) remain the most lethal malignancies amongst women worldwide and the second leading cause of cancer-related mortalities in the US. Subtype heterogeneity and aggressive invasive potential are believed to be the major contributors of these outcomes. BC lacking canonical histological receptors (i.e. ER/PR/HER2), called triple-negative (TNBC), are notoriously aggressive, difficult-to-treat, and metastatic. It has been observed that the degree of inflammation-driven tumorigenesis tends to correlate with increased levels of cell-free DNA (cfDNA) in cancer patient sera. Our lab had previously shown that nucleic-acid scavengers (NASs) could be used to block the pro-inflammatory and pro-invasive/metastatic signals (e.g. DAMPs) elicited by these cfDNA/RNA likely through innate immune sensors such as of the toll-like receptors (TLRs). Recently, we showed that treatment with the cationic polymer NAS, PAMAM-G3, elicited a drastic reduction in metastatic tumor burden to the liver in an immune-competent murine model of pancreatic cancer (I. Naqvi & R. Gunaratne et al., Molecular Therapy 26, 2018). Ongoing work has shown that both chemotherapy-derived TNBC conditioned media and a TLR9 agonist greatly increased TNBC in vitro invasion and was significantly inhibited upon treatment with PAMAM-G3. To elucidate the mechanism by which this NAS works in these tumor settings, our lab has developed several PAMAM-G3 derivatives, including biotin, IR-, and near-IR fluorophore labeled molecules. These molecules will allow us to conduct DAMP capture and characterization experiments, as well as perform in vitro and in vivo live imaging experiments to gain better insights into NAS PK/PD properties. Mechanistic insight into NAS anti-metastatic and anti-inflammatory capabilities will enhance our basic understanding of metastatic progression and its interplay with the immune system. Moreover, these principles will aid in the development of novel of anti-metastatic therapies to improve TNBC patient outcomes. Citation Format: Elias O. Eteshola, Ibtehaj A. Naqvi, Ruwan Gunaratne, Angelo Moreno, Smita K. Nair, Bruce A. Sullenger. Utilizing nucleic-acid scavengers (NASs) to inhibit proinflammatory and proinvasive signaling in triple-negative breast cancer [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 4515.

Abstract 5221: Snail promotes neurite outgrowth in prostate cancer cells

Abstract Neurite Outgrowth is a process where developing neurons produce new tentacle-like extensions as they grow in response to guidance cues. The projection can be an axon or a dendrite (nerve fibers). Nerve growth factors, or neurotrophins which are important for survival or growth, are one family of such stimuli that regulate neurite growth. Studies have also proposed that cancer cells stimulate their own innervations. Therefore, the concept of neoneurogenesis includes the development of nerve endings (axons)towards the tumor. Cancer cells are able to secrete neurite outgrowth-promoting molecules and axon guidance molecules that would stimulate and initiate the growth of these new axons to particular areas of the tumor. Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the peripheral nerve environment. Neurite Outgrowth could serve as a possible mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail1 or Snail is a zinc-finger protein that can down-regulate cell adhesion proteins such as E-cadherin by binding several E-boxes located in the promotor region, thereby inducing the epithelial mesenchymal transition (EMT) process to promote tumor migration and metastasis. Snail has also been shown to promote neuroendocrine differentiation. The hypothesis is that Snail can promote neurite outgrowth. For this study we utilized various prostate cancer cell lines: C42 NS, E006AA-hT (controls); C42 Snail shRNA, E006AA-hT (stable Snail knockdowns); LNCAP Neo (empty vector control) and LNCAP Snail (stably over-expressing Snail). Conditioned media collected from these cells was cultured with nerve cells (NS20Y) for 48 hours followed by a quantitative neurite outgrowth assay. Nerve growth factor (NGF) was utilized as a positive control. We also included Lanreotide, an inhibitor of neuroendocrine differentiation. After 48 hours, the nerve cells were incubated with Fix/Stain solution containing Cell Viability, and Cell Membrane stain, and nerve extensions quantified by analysis with a plate reader. Our results showed that C42 NS and E006AA-hT NS (Snail-high) conditioned media led to a higher neurite outgrowth compared to the Snail knocked down cells. We also observed that neurite outgrowth was blocked by Lanreotide inhibitor. In conclusion, Snail promotes neurite outgrowth via secretion of soluble factors, which can be inhibited by Lanreotide, a drug currently being used to treat patients with neuroendocrine tumors. Therefore, targeting cancer cell interaction with nerve cells may be helpful in halting prostate cancer progression/metastasis. GRANT SUPPORT: NIH 1P20MD002285, NIH/NCRR/RCMI G12RR003062-22 and NIH/NIGMS/RISE 5R25GM060414 Citation Format: Gabrielle J. Edwards. Snail promotes neurite outgrowth in prostate cancer cells [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 5221.

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.

PYK2 promotes HER2-positive breast cancer invasion

BackgroundMetformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer.MethodsThe effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER−/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER−/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER−/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER−/PR- patients’ cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER−/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays.ResultsOur study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44.ConclusionsWe provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients.

Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells.

Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl–Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl–Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling.

Going off the grid: ERα breast cancer beyond estradiol.

Novel studies have linked cholesterol biosynthesis to drug resistance in luminal breast cancer. Structural data suggest that cholesterol metabolites, including 27-hydroxycholesterol (27HC), can act as ERα ligands in these cells. Additionally, hypercholesterolemia has now been linked to breast cancer progression. The focus of this review is to briefly summarize these recent findings and discuss how epigenetic reprogramming is definitively connected to endogenous cholesterol biosynthesis. We elaborate on how these data support a working model in which cholesterol biosynthesis promotes autocrine, pro-invasive signaling via activation of a series of closely related transcription factors. Importantly, we discuss how this mechanism of resistance is specifically associated with aromatase inhibitors. Finally, we examine how the field is now considering the development of anticholesterol therapeutics and companion biomarkers to stratify and treat ERα breast cancer patients. In particular, we review recent progress in pharmaceutical strategies targeting the cholesterol molecular machinery in primary and secondary breast cancers.

Co-option of Liver Vessels and Not Sprouting Angiogenesis Drives Acquired Sorafenib Resistance in Hepatocellular Carcinoma.

Background: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is ‘vessel co-option,’ ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents.Methods: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology.Results: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic ‘rebound’ effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option.Conclusions: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling.

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice.

During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.

Neutralization of the CD95 ligand by APG101 inhibits invasion of glioma cells in vitro.

Glioblastoma is a disease characterized by rapid invasive tumour growth. Studies on the proapoptotic CD95/CD95L signalling pathway recently suggested a significant contribution of CD95 signalling towards the high degree of motility in glioma cells. Apogenix has developed APG101, a clinical phase II compound designed to bind and neutralize CD95L, and thus to interfere with CD95/CD95L-based signalling. APG101 has shown clinical efficacy in a controlled randomized phase II trial in patients with recurrent glioma. Because APG101 is not cytotoxic to tumour cells in vitro, we postulated that the anti-invasive function of APG101 is the main mechanism of action for this compound. Using three-dimensional spheroid invasion assays in vitro and in murine brain tissue cultures, we found that knockdown of endogenous CD95L reduced the invasive phenotype in our two glioblastoma model cell lines U87-MG and U251-MG. Invasion was restored in CD95L knockdown cells upon the addition of soluble recombinant CD95L and this effect was inhibited by APG101. We conclude that CD95L from autocrine and paracrine sources contributes towards the invasive phenotype of glioblastoma cells and that APG101 acts as a suppressor of proinvasive signalling by the CD95/CD95L pathway in glioblastoma.

Gap junctions modulate glioma invasion by direct transfer of microRNA.

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.

Abstract 3897: Sam68 sustains self-renewal and invasiveness of breast cancer initiating cells

Abstract Background: Breast cancer is the leading worldwide cause of death among women due to the high metastatic spread of this disease. As by definition, Cancer Initiating Cells (CICs) are a fraction of primary tumor cells harboring tumorigenic potential and successful outgrowth at metastatic sites. Targeting molecular events affecting CICs peculiarities, as self-renewal and an innate chemoresistance, could improve the ineffectiveness of current therapies. Sam68, the major CD44 splicing regulator, is a multifunctional RNA-binding protein involved in multiple steps of RNA metabolism and its expression is aberrant in breast cancer. Herein, we highlight novel implications of Sam68 in the mammary tumor onset and progression. Material and methods: Tissue microarrays (TMA) were composed of n=155 histologically confirmed invasive breast carcinoma (T1-T2, N0, M0) and Sam68 expression was quantified as percent of immunoreactive tumor cells, a cut-off of 90% positive cells was chosen. Breast CICs were obtained from mechanically and enzimatically digestion of human breast tissues. Stable Sam68 knockdown was produced by lentiviral transduction and puromicin selection. Invasion assay was performed in 8µm pore size transwell coated with GF-depleted matrigel 1:3 in serum free medium. 3x104 cells were plated and 10% FBS medium was used as chemoattractant. Breast CICs (3 x 105) were suspended in 1:6 matrigel and orthotopically injected in NOD/SCID mice. Results: Univariate analysis of the TMA data showed that invasive breast carcinoma over-expressing Sam68 (≥ 90% positive cells) were associated with a significantly higher incidence of distant relapse (P = 0.011). Notably, in a cohort of screened breast cancer patients, breast CICs expressed higher Sam68 protein levels than tumor bulk, highlighting that Sam68 expression was likely restricted to cells with self-renewal activity. Stable knockdown of Sam68 in breast CICs significantly curtailed proliferation rate and was coupled with an increase in p27kip1 and reduction of Bcl-xL. Compared to control cells, downregulation of Sam68 was correlated with an attenuation of cell motility capability and Twist, Snail, CD44v6 and Met levels. Moreover, Sam68 modulated the expression of an oncogenic alternative splice variant of Met (Met-SM), probably through the ASF/SF2 splicing factor. In addition, depletion of Sam68 sensitized breast CICs to standard chemotherapy. Finally, Sam68 silencing ablated breast cancer xenograft formation in immunocompromised mice. Conclusions: Based on these results, we deem that Sam68 may promote pro-invasive signals by inducing and modulating the alternative splicing of oncogenic variants of several genes, such as CD44 and Met. Thus, further investigations on signaling pathways affecting CICs self-renewal and invasiveness could represent a crucial key to improve selective cancer treatment. Citation Format: Alice Turdo, Miriam Gaggianesi, Antonina Benfante, Mauro Piantelli, Matilde Todaro, Giorgio Stassi. Sam68 sustains self-renewal and invasiveness of breast cancer initiating cells. [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 3897. doi:10.1158/1538-7445.AM2014-3897