Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (> 50) have implicated single nucleotide polymorphisms (SNPs) located at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of breast cancer. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for breast cancer susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, account for GWAS-associated breast cancer risk at this locus. Breast cancer-associated CCDC170 nonsense mutations and rearrangements have also been detected, with the latter being specifically implicated in driving breast cancer. Here we report that the wild type CCDC170 protein localizes to the region of the Golgi apparatus and binds Golgi-associated microtubules (MTs), and that breast cancer-linked truncations of CCDC170 result in loss of Golgi localization. Overexpression of wild type CCDC170 triggers Golgi reorganization, and enhances Golgi-associated MT stabilization and acetyltransferase ATAT1-dependent α-tubulin acetylation. Golgi-derived MTs regulate cellular polarity and motility, and we provide evidence that dysregulation of CCDC170 affects polarized cell migration. Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and implicate a mechanism for how perturbations in the CCDC170 gene may contribute to the hallmark changes in cell polarity and motility seen in breast cancer.

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The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of>30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

Background: Breast cancer (BCa) is a genetically heterogeneous disease and many genes contributing to BCa risk remain to be identified. Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (>50) have strongly implicated genetic alterations at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of BCa. ESR1, encoding the estrogen receptor, might be a more obvious candidate for influencing risk. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for BCa susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, accounts for GWAS-associated BCa risk at this locus. CCDC170 is a coiled-coiled domain-containing protein of poorly understood function. BCa-specific truncation and missense mutations in CCDC170 also have been detected, with the truncations being implicated in driving Luminal B subtype BCa. Together these findings demonstrate that the CCDC170 gene is involved in BCa, but the underlying molecular mechanisms for its role in tumorigenesis are unknown. Experimental designs and results: By using the approaches of confocal microscopy and cell imaging analysis, here we report for the first time that CCDC170 is associated with the Golgi apparatus and perinuclear microtubules (MTs), and support a role for CCDC170 in the Golgi-associated microtubule network. We have shown that overexpression of CCDC170 triggers Golgi reorganization and stabilizes Golgi-associated MTs, accompanied by dramatically increased acetylation of α-tubulin that is driven by the acetyltransferase ATAT1. The Golgi-associated MT network has been proposed to regulate cell polarity and migration. In support of this concept, we have shown that CRISPR knockout increases, and overexpression of CCDC170 decreases, BCa directional cell migration in vitro. We also found that the BCa-specific truncations result in mislocalization of CCDC170 and/or diminished stability of Golgi-associated MTs. Lastly, we identified candidate CCDC170 functional binding partners (e.g. MAP4) that are consistent with its localization and proposed function. These partners may serve to mediate the acetylation and stabilization of MTs. Conclusions: Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and provide a mechanism for how perturbations in CCDC170 could alter Golgi-mediated cell polarity, and thereby drive BCa and other abnormalities. This work was partially supported by the Susan G. Komen for the Cure (KG100274), NCI (CA186853), and Eileen Stein Jacoby Fund. Citation Format: Pengtao Jiang, Yueran Li, Andrey Poleshko, Valentina Medvedeva, Natalia Baulina, Yongchao Zhang, Yan Zhou, Carolyn M. Slater, Trinity Pellegrin, Jason Wasserman, Michael Lindy, Mary Daly, Richard A. Katz, Xiaowei Chen. The breast cancer gene CCDC170 regulates the Golgi-associated microtubule network and directional cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1884. doi:10.1158/1538-7445.AM2017-1884

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