Fate and Efficacy of Engineered Allogeneic Stem Cells Targeting Cell Death and Proliferation Pathways in Primary and Brain Metastatic Lung Cancer.

Primary and metastatic lung cancer is a leading cause of cancer-related death and novel therapies are urgently needed. Epidermal growth factor receptor (EGFR) and death receptor (DR) 4/5 are both highly expressed in primary and metastatic non-small cell lung cancer (NSCLC); however, targeting these receptors individually has demonstrated limited therapeutic benefit in patients. In this study, we created and characterized diagnostic and therapeutic stem cells (SC), expressing EGFR-targeted nanobody (EV) fused to the extracellular domain of death DR4/5 ligand (DRL) (EVDRL) that simultaneously targets EGFR and DR4/5, in primary and metastatic NSCLC tumor models. We show that EVDRL targets both cell surface receptors, and induces caspase-mediated apoptosis in a broad spectrum of NSCLC cell lines. Utilizing real-time dual imaging and correlative immunohistochemistry, we show that allogeneic SCs home to tumors and when engineered to express EVDRL, alleviate tumor burden and significantly increase survival in primary and brain metastatic NSCLC. This study reports mechanistic insights into simultaneous targeting of EGFR- and DR4/5 in lung tumors and presents a promising approach for translation into the clinical setting.

The impact of synchronous versus asynchronous electrical stimulation in artificial vision.

Visual prosthesis devices designed to restore sight to the blind have been under development in the laboratory for several decades. Clinical translation continues to be challenging, due in part to gaps in our understanding of critical parameters such as how phosphenes, the electrically-generated pixels of artificial vision, can be combined to form images. In this review we explore the effects that synchronous and asynchronous electrical stimulation across multiple electrodes have in evoking phosphenes. Understanding how electrical patterns influence phosphene generation to control object binding and perception of visual form is fundamental to creation of a clinically successful prosthesis.

Anti-EGFR VHH-armed death receptor ligand-engineered allogeneic stem cells have therapeutic efficacy in diverse brain metastatic breast cancers.

Basal-like breast cancer (BLBC) shows brain metastatic (BM) capability and overexpresses EGFR and death-receptors 4/5 (DR4/5); however, the anatomical location of BM prohibits efficient drug-delivery to these targetable markers. In this study, we developed BLBC-BM mouse models featuring different patterns of BMs and explored the versatility of estem cell (SC)-mediated bi-functional EGFR and DR4/5-targeted treatment in these models. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, EVDRL [anti-EGFR VHH (EV) fused to DR ligand (DRL)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the EV domain facilitated in augmenting DR4/5-DRL binding and enhancing DRL-induced apoptosis. EVDRL secreting stem cells alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. This study reports mechanism based simultaneous targeting of EGFR and DR4/5 in BLBC and defines a new treatment paradigm for treatment of BM.

Regulation of localization and function of the transcriptional co-activator YAP by angiomotin.

The Hippo-YAP pathway is a central regulator of cell contact inhibition, proliferation and death. There are conflicting reports regarding the role of Angiomotin (Amot) in regulating this pathway. While some studies suggest a YAP-inhibitory function other studies indicate Amot is required for YAP activity. Here, we describe an Amot-dependent complex comprised of Amot, YAP and Merlin. The phosphorylation of Amot at Serine 176 shifts localization of this complex to the plasma membrane, where it associates with the tight-junction proteins Pals1/PATJ and E-cadherin. Conversely, hypophosphorylated Amot shifts localization of the complex to the nucleus, where it facilitates the association of YAP and TEAD, induces transcriptional activation of YAP target genes and promotes YAP-dependent cell proliferation. We propose that phosphorylation of AmotS176 is a critical post-translational modification that suppresses YAP's ability to promote cell proliferation and tumorigenesis by altering the subcellular localization of an essential YAP co-factor.

Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1.

Neurofibromatosis type 2 (NF2) is a dominantly inherited autosomal disease characterized by schwannomas of the 8th cranial nerve. The NF2 tumor suppressor gene encodes for Merlin, a protein implicated as a suppressor of multiple cellular signaling pathways. To identify potential drug targets in NF2-associated malignancies we assessed the consequences of inhibiting the tyrosine kinase receptor MET. We identified crizotinib, a MET and ALK inhibitor, as a potent inhibitor of NF2-null Schwann cell proliferation in vitro and tumor growth in vivo. To identify the target/s of crizotnib we employed activity-based protein profiling (ABPP), leading to identification of FAK1 (PTK2) as the relevant target of crizotinib inhibition in NF2-null schwannoma cells. Subsequent studies confirm that inhibition of FAK1 is sufficient to suppress tumorigenesis in animal models of NF2 and that crizotinib-resistant forms of FAK1 can rescue the effects of treatment. These studies identify a FDA approved drug as a potential treatment for NF2 and delineate the mechanism of action in NF2-null Schwann cells.

Crumbs 3b promotes tight junctions in an ezrin-dependent manner in mammalian cells.

Crumbs 3 (CRB3) is a component of epithelial junctions, which has been implicated in apical-basal polarity, apical identity, apical stability, cell adhesion, and cell growth. CRB3 undergoes alternative splicing to yield two variants: CRB3a and CRB3b. Here, we describe novel data demonstrating that, as with previous studies on CRB3a, CRB3b also promotes the formation of tight junctions (TJs). However, significantly we demonstrate that the 4.1-ezrin-radixin-moesin-binding motif of CRB3b is required for CRB3b functionality and that ezrin binds to the FBM of CRB3b. Furthermore, we show that ezrin contributes to CRB3b functionality and the correct distribution of TJ proteins. We demonstrate that both CRB3 isoforms are required for the production of functionally mature TJs and also the localization of ezrin to the plasma membrane. Finally, we demonstrate that reduced CRB3b expression in head and neck squamous cell carcinoma (HNSCC) correlates with cytoplasmic ezrin, a biomarker for aggressive disease, and shows evidence that while CRB3a expression has no effect, low CRB3b and high cytoplasmic ezrin expression combined may be prognostic for HNSCC.

Fibroblasts in Head and Neck Squamous Cell Carcinoma Associated With Perineural Invasion Have High-Level Nuclear Yes-Associated Protein (YAP) Expression.

We retrospectively studied the expression of Yes-associated protein (YAP) using immunohistochemical staining in 10 cases of head and neck squamous cell carcinoma with associated perineural invasion. We find that fibroblasts in areas associated with perineural invasion show higher levels of nuclear YAP compared to fibroblasts in the stroma of normal mucosa, with a median cell count of 35.4 per high-power field in the former and 3.9 in the latter. No differences were observed between the expression of YAP phosphorylated at Ser127 in the tumoral stroma compared to that in the normal mucosa, with a median cell count expression of 4.9 in the former versus 5.0 in the latter. Therefore, a strong and increased nuclear YAP expression in fibroblasts associated with perineural invasion in head and neck squamous cell carcinoma suggests that YAP-mediated transcription programs in these fibroblasts may contribute to perineural invasion.

The Angiomotins--from discovery to function.

Angiomotins were originally identified as angiostatin binding proteins and implicated in the regulation of endothelial cell migration. Recent studies have shed light on the role of Angiomotins and other members of the Motin protein family in epithelial cells and in pathways directly linked to the pathogenesis of cancer. In particular, Motins have been shown to play a role in signaling pathways regulated by small G-proteins and the Hippo-YAP pathway. In this review the role of the Motin protein family in these signaling pathways will be described and open questions will be discussed.

The expanding family of FERM proteins.

Our understanding of the FERM (4.1/ezrin/radixin/moesin) protein family has been rapidly expanding in the last few years, with the result that many new physiological functions have been ascribed to these biochemically unique proteins. In the present review, we will discuss a number of new FRMD (FERM domain)-containing proteins that were initially discovered from genome sequencing but are now being established through biochemical and genetic studies to be involved both in normal cellular processes, but are also associated with a variety of human diseases.

Willin, an upstream component of the hippo signaling pathway, orchestrates mammalian peripheral nerve fibroblasts.

Willin/FRMD6 was first identified in the rat sciatic nerve, which is composed of neurons, Schwann cells, and fibroblasts. Willin is an upstream component of the Hippo signaling pathway, which results in the inactivation of the transcriptional co-activator YAP through Ser127 phosphorylation. This in turn suppresses the expression of genes involved in cell growth, proliferation and cancer development ensuring the control of organ size, cell contact inhibition and apoptosis. Here we show that in the mammalian sciatic nerve, Willin is predominantly expressed in fibroblasts and that Willin expression activates the Hippo signaling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These results show that Willin modulates sciatic nerve fibroblast activity indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.