HER-Family Ligands Promote Acquired Resistance to Trastuzumab in Gastric Cancer.

Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line-based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer.

An Integrative Study of Protein-RNA Condensates Identifies Scaffolding RNAs and Reveals Players in Fragile X-Associated Tremor/Ataxia Syndrome.

Recent evidence indicates that specific RNAs promote the formation of ribonucleoprotein condensates by acting as scaffolds for RNA-binding proteins (RBPs). We systematically investigated RNA-RBP interaction networks to understand ribonucleoprotein assembly. We found that highly contacted RNAs are structured, have long UTRs, and contain nucleotide repeat expansions. Among the RNAs with such properties, we identified the FMR1 3' UTR that harbors CGG expansions implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). We studied FMR1 binding partners in silico and in vitro and prioritized the splicing regulator TRA2A for further characterization. In a FXTAS cellular model, we validated the TRA2A-FMR1 interaction and investigated implications of its sequestration at both transcriptomic and post-transcriptomic levels. We found that TRA2A co-aggregates with FMR1 in a FXTAS mouse model and in post-mortem human samples. Our integrative study identifies key components of ribonucleoprotein aggregates, providing links to neurodegenerative disease and allowing the discovery of therapeutic targets.

The First-in-class Anti-EGFR Antibody Mixture Sym004 Overcomes Cetuximab Resistance Mediated by EGFR Extracellular Domain Mutations in Colorectal Cancer.

PURPOSE: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. EXPERIMENTAL DESIGN: Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. RESULTS: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. CONCLUSIONS: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260-7. ©2016 AACR.

Dynamics of KRas on endosomes: involvement of acidic phospholipids in its association.

The endocytic compartment is emerging as a functional platform for controlling important cellular processes. We have found that ∼10 to 15% of total KRas, a protein that is frequently mutated in cancer, is present on endosomes, independent of its activation state. The dynamics of GFP-KRas wild-type (WT) and constitutively active or inactive mutants on endosomes were analyzed by fluorescence recovery after photobleaching (FRAP) microscopy. The measurements revealed an extraordinarily fast recovery of KRas WT [half-time (HT), ∼1.3 s] compared to HRas, Rab5, and EGFR, with the active KRasG12V mutant being significantly faster and more mobile (HT, ∼1 s, and ∼82% of exchangeable fraction) than the inactive KRasS17N (HT, ∼1.6 s, and ∼60% of exchangeable fraction). KRas rapidly switches from the cytoplasm to the endosomal membranes by an electrostatic interaction between its polybasic region and the endosomal acidic phospholipids, mainly phosphatidylserine.-Gelabert-Baldrich, M., Soriano-Castell, D., Calvo, M., Lu, A., Viña-Vilaseca, A., Rentero, C., Pol, A., Grinstein, S. Enrich, C., Tebar, F. Dynamics of KRas on endosomes: involvement of acidic phospholipids in its association.

Annexins and endosomal signaling.

Cell signaling and endocytosis are intimately linked in eukaryotic cells. Signaling receptors at the cell surface enter the endocytic pathway and continue to activate downstream effectors in endosomal compartments. This spatiotemporal regulation of signal transduction provides opportunity for signal diversity and a cell-specific machinery of scaffolding/targeting proteins contributes to establish compartment-specific signaling complexes. Members of the annexin (Anx) protein family, in particular AnxA1, AnxA2, and AnxA6, appear to target their interaction partners to specific membrane microdomains to contribute to the formation of compartment-specific signaling platforms along the endocytic pathway. A major challenge to understand the impact of scaffolding/targeting proteins on spatiotemporal signal transduction along endocytic pathways is the identification, isolation, and functional analysis of low-abundance signal-transducing protein complexes in endocytic compartments. Here, we describe methods to isolate endosomes and to target signaling molecules to endosomes. Applying these methodologies to suitable animal or cell models will enable the dissection of signal transduction in the endocytic compartment in the presence or absence of annexins.

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains.

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of "emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.

Rac1 and calmodulin interactions modulate dynamics of ARF6-dependent endocytosis.

The main cellular Ca(2+) sensor, calmodulin (CaM), interacts with and regulates several small GTPases, including Rac1. The present study revealed high binding affinity of Rac1 for CaM and uncovered two new essential binding domains in Rac1: the polybasic region, important for phosphatidylinositol-4-phosphate 5-kinase (PIP5K) interaction, and the adjacent prenyl group. CaM inhibition increased Rac1 binding to PIP5K and induced an extensive phosphatidylinositol 4,5-bisphosphate (PI4,5P(2) )-positive tubular membrane network. Immunofluorescence demonstrated that the tubules were plasma membrane invaginations resulting from an ADP-ribosylation factor 6 (ARF6)-dependent and clathrin-independent pathway. The role of Rac1 in this endocytic route was analyzed by expressing constitutively active and inactive mutants. While active Rac1 impaired tubulation, the inactive mutant enhanced it. Intriguingly, inactive mutant expression elicited tubulation by recruiting PIP5K and inhibiting Rac1 at the plasma membrane. Accordingly, CaM inhibition inactivated Rac1 and increased Rac1/PIP5K interaction. Therefore, our findings highlight an important new role for Rac1 and CaM in controlling clathrin-independent endocytosis.