Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti-ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.T he epidermal growth factor related protein (ErbB) family of receptor tyrosine kinases plays an important role in epitheliogenesis and, accordingly, serves as a major therapeutic target in several cancers. The family comprises four transmembrane receptors and 11 ligands that induce homodimerization or heterodimerization upon binding to the respective receptor (1). ErbB-1 (also called the epidermal growth factor receptor; EGFR) and ErbB-4 share some ligands, whereas no similar ligand is so far known for ErbB-2. Overexpression and mutations of ErbB family members lead to a multitude of malignancies. To date, synthetic tyrosine kinase inhibitors (e.g., Erlotinib and Gefitinib), as well as monoclonal antibodies (mAbs; e.g., Cetuximab and Trastuzumab), have been developed to inhibit pathological signaling or recruit the immune system to cancer cells (2). Aptamers might represent an alternative therapeutic modality. These molecules are small, singlestranded DNA or RNA molecules (3). RNA aptamers were described for the first time in 1990 by two laboratories (4, 5). Since then, aptamers against a multitude of different organic and inorganic, small and macromolecular, targets were developed. In addition, high-affinity aptamer binding ranging from picomolar to low nanomolar concentrations have been documented (6). Aptamers are selected in an evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). A DNA or RNA library containing single-stranded random ...

Epidermal growth factor receptor (EGFR) mutations identify patients with lung cancer who derive benefit from kinase inhibitors. However, most patients eventually develop resistance, primarily due to the T790M second‐site mutation. Irreversible inhibitors (e.g., osimertinib/AZD9291) inhibit T790M‐EGFR, but several mechanisms, including a third‐site mutation, C797S, confer renewed resistance. We previously reported that a triple mixture of monoclonal antibodies, 3×mAbs, simultaneously targeting EGFR, HER2, and HER3, inhibits T790M‐expressing tumors. We now report that 3×mAbs, including a triplet containing cetuximab and trastuzumab, inhibits C797S‐expressing tumors. Unlike osimertinib, which induces apoptosis, 3×mAbs promotes degradation of the three receptors and induces cellular senescence. Consistent with distinct mechanisms, treatments combining 3×mAbs plus sub‐inhibitory doses of osimertinib synergistically and persistently eliminated tumors. Thus, oligoclonal antibodies, either alone or in combination with kinase inhibitors, might preempt repeated cycles of treatment and rapid emergence of resistance.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Because of emergence of resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), no targeted treatments are available for patients with lung cancer who lose sensitivity due to new mutations or bypass mechanisms. We examined in animals and an alternative therapeutic approach making use of antibodies. An osimertinib-sensitive animal model of lung cancer, which rapidly develops drug resistance, has been employed. To overcome compensatory hyperactivation of ERK, which we previously reported, an anti-EGFR antibody (cetuximab) was combined with other antibodies, as well as with a subtherapeutic dose of osimertinib, and cancer cell apoptosis was assayed. Our animal studies identified a combination of three clinically approved drugs, cetuximab, trastuzumab (an anti-HER2 mAb), and osimertinib (low dose), as an effective and long-lasting treatment that is able to prevent onset of resistance to osimertinib. A continuous schedule of concurrent treatment was sufficient for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumor extracts indicated that the combination of two mAbs and a subtherapeutic TKI dose sorted EGFR and HER2 for degradation; cooperatively enhanced apoptosis; inhibited activation of ERK; and reduced abundance of several bypass proteins, namely MET, AXL, and HER3. Our assays and animal studies identified an effective combination of clinically approved drugs that might overcome resistance to irreversible TKIs in clinical settings. The results we present attribute the long-lasting effect of the drug combination to simultaneous blockade of several well-characterized mechanisms of drug resistance. .

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.