Abstract P6-10-04: The Presence of Anaplastic Lymphoma Kinase Recapitulates Formation of Breast Tumor Emboli with Encircling Lymphovasculogenesis
Authors: Liu H , Chu K , Ochoa AE , Ye Z , Zhang X , Jin J , Wright MC , Barsky SH , Cristofanilli M , Robertson FM 
Published: August 2014

Abstract
Background: Genetic abnormalities in the anaplastic lymphoma kinase (ALK) gene result in activation of signaling pathways including Akt, mTor, and JAK/Stat3. ALK has been shown to be a primary oncogenic driver in a variety of human tumors, including both hematologic malignancies such as anaplastic large cell lymphoma, as well as solid tumors including neuroblastoma, non-small cell lung cancer, myofibroblastic tumors and most recently, high grade serous ovarian carcinoma. While only ∼3% of all breast cancers have been reported to have ALK genetic abnormalities, our studies revealed that inflammatory breast cancer (IBC), the most lethal variant of breast cancer, is characterized by prevalent ALK gene amplification with activated ALK signaling. The present studies investigated the role of ALK in breast cancer by expressing full-length wild type ALK in MCF-7 cells.
Materials and Methods: Clones of MCF-7 breast cancer cells expressing wild type ALK or non-target vector were produced by lentivirus infection and selection of single cell clones. MCF-7 ALK clones were evaluated using live cell and phase contrast imaging, immunofluorescent staining with confocal imaging, gene profiling, phospho-protein array analysis, western blot and ELISA validation. In vivo studies were performed by injection of MCF-7 ALK clones into using NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice using IACUC approved animal protocols.
Results: When cultured on plastic substrates, MCF-7 ALK clones formed tumor cell aggregates instead of monolayer cultures, and when cultured as tumor spheroids under non-adherent 3D conditions had a distinct cellular phenotype with significantly greater clonogenicity than either non-target vector MCF-7 clones or the parental cell line. Whole transcriptome analysis, with validation using protein arrays, western blots and ELISA analysis revealed that the presence of ALK up-regulated phospho-src. In vivo studies revealed that ALK expressing MCF-7 clones formed tumor emboli that were enwrapped by dermal lymphatic vessels, essentially recapitulating the phenotype of IBC tumor emboli that exhibit encircling lymphovasculogenesis. Enforced expression of wild type ALK in another breast cancer cell line resulted in similar formation of tumor emboli.
Discussion: These studies provide first time evidence for the association between full length ALK and formation of highly invasive tumor emboli enwrapped by lymphatic vessels, which is a primary characteristic of IBC. These studies, taken together with discovery of the prevalence of ALK gene amplification in IBC patients, indicate that ALK represents an important therapeutic target for IBC, with the availability of new ALK targeted therapies to evaluate as single agents and in combinations with other agents that may effectively target IBC tumor emboli that we have now linked to ALK and which represent the metastatic lesion of this lethal variant of breast cancer.
Funding by Susan G. Komen Organization Promise Grant KG081287 (FMR and MC).
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-04.