The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.ion channel | CaCCinh-A01 | TMEM16A | HNSCC | ESCC

We performed a focused siRNA screen in an A549 dengue type 2 New Guinea C subgenomic replicon cell line (Rluc-replicon) that contains a Renilla luciferase cassette. We found that siRNA mediated knock down of mevalonate diphospho decarboxylase (MVD) inhibited viral replication of the Rluc-replicon and DEN-2 NGC live virus replication in A549 cells. When the Rluc-replicon A459 cells were grown in delipidated media the replicon expression was suppressed and MVD knock down could further sensitize Renilla expression. Hymeglusin and zaragozic acid A could inhibit DEN-2 NGC live virus replication in K562 cells, while lovastatin could inhibit DEN-2 NGC live virus replication in human peripheral blood mononuclear cells. Renilla expression could be rescued in fluvastatin treated A549 Rluc-replicon cells after the addition of mevalonate, and partially restored with geranylgeranyl pyrophosphate, or farnesyl pyrophosphate. Our data suggest genetic and pharmacological modulation of cholesterol biosynthesis can regulate dengue virus replication.

Background: The calcium-activated chloride channel ANO1 is highly expressed in cancer.Results: Inhibition of ANO1 activity alone is not sufficient to inhibit cancer cell proliferation, suggesting a novel function of ANO1 protein in cancer.Conclusion: The ANO1 inhibitor CaCCinh-A01 inhibits cancer cell proliferation by facilitating degradation of ANO1.Significance: Our results may provide a new targeting approach for antitumor therapy in ANO1-amplified cancers.

Mature lymphomas produced in Rhode Island

Tumor types can be defined cytologically by their regions of chromosomal amplification, which often results in the high expression of both mRNA and proteins of certain genes contained within the amplicon. An important strategy for defining therapeutically relevant targets in these situations is to ascertain which genes are amplified at the protein level and, concomitantly, are key drivers for tumor growth or maintenance. Furthermore, so-called passenger genes that are amplified with driver genes and a manifest on the cell surface can be attractive targets for an antibody-drug conjugate approach (ADC). We employed a tandem mass spectrometry proteomics approach using tumor cell lines to identify the cell surface proteins whose expression correlates with the 11q13 amplicon. The 11q13 amplicon is one of the most frequently amplified chromosomal regions in human cancer, being present in 45% of head and neck and oral squamous cell carcinoma (OSCC) and 13-21% of breast and liver carcinomas. Using a panel of tumor cell lines with defined 11q13 genomic amplification, we identified the membrane proteins that are differentially expressed in an 11q13 amplified cell line panel using membrane-enriched proteomic profiling. We found that DSG3, CD109, and CD14 were differentially overexpressed in head and neck and breast tumor cells with 11q13 amplification. The level of protein expression of each gene was confirmed by Western blot and FACS analysis. Because proteins with high cell surface expression on selected tumor cells could be potential antibody drug conjugate targets, we tested DSG3 and CD109 in antibody piggyback assays and validated that DSG3 and CD109 expression was sufficient to induce antibody internalization and cell killing in 11q13-amplified cell lines. Our results suggest that proteomic profiling using genetically stratified tumors can identify candidate antibody drug conjugate targets. Data are available via ProteomeXchange with the identifier PXD002486.