Cytokeratins 5 and 17 Maintain an Aggressive Epithelial State in Basal-Like Breast Cancer.

Basal-like breast cancers (BLBC) are the most common triple-negative subtype (hormone receptor and HER2 negative) with poor short-term disease outcome and are commonly identified by expression of basal cytokeratins (CK) 5 and 17. The goal of this study was to investigate whether CK5 and CK17 play a role in adverse behavior of BLBC cells. BLBC cell lines contain heterogeneous populations of cells expressing CK5, CK17, and the mesenchymal filament protein vimentin. Stable shRNA knockdown of either CK5 or CK17 compared with non-targeting control in BLBC cells was sufficient to promote an epithelial-mesenchymal transition (EMT) gene signature with loss of E-cadherin and an increase in vimentin expression. Relative to control cells, CK5 and CK17 knockdown cells acquired a more spindle-like morphology with increased cell scattering and were more invasive in vitro. However, CK5 or CK17 knockdown compared with control cells generated decreased lymph node and lung metastases in vivo. Loss of CK5 or CK17 moderately reduced the IC50 dose of doxorubicin in vitro and led to increased doxorubicin efficacy in vivo. Single-cell RNA-sequencing of BLBC patient-derived xenografts identified heterogeneous populations of CK5/CK17, vimentin, and dual basal CK/vimentin-positive cells that fell on an EMT spectrum of epithelial, mesenchymal, and intermediate, respectively, whereas knockdown of CK5 transitioned cells toward a more mesenchymal score. IMPLICATIONS: This study supports that basal CKs 5 and 17 contribute to the adverse behavior of BLBC cells and could be an untapped source of therapeutic vulnerability for this aggressive disease.

Estrogens and Progestins Cooperatively Shift Breast Cancer Cell Metabolism.

Metabolic reprogramming remains largely understudied in relation to hormones in estrogen receptor (ER) and progesterone receptor (PR) positive breast cancer. In this study, we investigated how estrogens, progestins, or the combination, impact metabolism in three ER and PR positive breast cancer cell lines. We measured metabolites in the treated cells using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Top metabolic processes upregulated with each treatment involved glucose metabolism, including Warburg effect/glycolysis, gluconeogenesis, and the pentose phosphate pathway. RNA-sequencing and pathway analysis on two of the cell lines treated with the same hormones, found estrogens target oncogenes, such as MYC and PI3K/AKT/mTOR that control tumor metabolism, while progestins increased genes associated with fatty acid metabolism, and the estrogen/progestin combination additionally increased glycolysis. Phenotypic analysis of cell energy metabolism found that glycolysis was the primary hormonal target, particularly for the progestin and estrogen-progestin combination. Transmission electron microscopy found that, compared to vehicle, estrogens elongated mitochondria, which was reversed by co-treatment with progestins. Progestins promoted lipid storage both alone and in combination with estrogen. These findings highlight the shift in breast cancer cell metabolism to a more glycolytic and lipogenic phenotype in response to combination hormone treatment, which may contribute to a more metabolically adaptive state for cell survival.

New generation breast cancer cell lines developed from patient-derived xenografts.

BACKGROUND: Breast cancer is a highly heterogeneous disease characterized by multiple histologic and molecular subtypes. While a myriad of breast cancer cell lines have been developed over the past 60 years, estrogen receptor alpha (ER)+ disease and some mutations associated with this subtype remain underrepresented. Here we describe six breast cancer cell lines derived from patient-derived xenografts (PDX) and their general characteristics. METHODS: Established breast cancer PDX were processed into cell suspensions and placed into standard 2D cell culture; six emerged into long-term passageable cell lines. Cell lines were assessed for protein expression of common luminal, basal, and mesenchymal markers, growth assessed in response to estrogens and endocrine therapies, and RNA-seq and oncogenomics testing performed to compare relative transcript levels and identify putative oncogenic drivers. RESULTS: Three cell lines express ER and two are also progesterone receptor (PR) positive; PAM50 subtyping identified one line as luminal A. One of the ER+PR+ lines harbors a D538G mutation in the gene for ER (ESR1), providing a natural model that contains this endocrine-resistant genotype. The third ER+PR-/low cell line has mucinous features, a rare histologic type of breast cancer. The three other lines are ER- and represent two basal-like and a mixed ductal/lobular breast cancer. The cell lines show varied responses to tamoxifen and fulvestrant, and three were demonstrated to regrow tumors in vivo. RNA sequencing confirms all cell lines are human and epithelial. Targeted oncogenomics testing confirmed the noted ESR1 mutation in addition to other mutations (i.e., PIK3CA, BRCA2, CCND1, NF1, TP53, MYC) and amplifications (i.e., FGFR1, FGFR3) frequently found in breast cancers. CONCLUSIONS: These new generation breast cancer cell lines add to the existing repository of breast cancer models, increase the number of ER+ lines, and provide a resource that can be genetically modified for studying several important clinical breast cancer features.

Cytokeratin 5 alters ß-catenin dynamics in breast cancer cells.

Estrogen receptor (ER) positive breast cancers often contain subpopulations of cells that express the intermediate filament protein cytokeratin 5 (CK5). CK5+ cells are enriched in cancer stem cell (CSC) properties, can be induced by progestins, and predict poor prognosis in ER+ breast cancer. We established through CK5 knockout and overexpression in ER+ breast cancer cell lines that CK5 is important for tumorsphere formation, prompting us to speculate that CK5 has regulatory activity in CSCs. To interrogate CK5 interacting proteins that may be functionally cooperative, we performed immunoprecipitation-mass spectrometry for CK5 in ER+ breast cancer cells. Focusing on proteins with signaling activity, we identified ß-catenin, a key transcription factor of the Wnt signaling pathway and cell adhesion molecule, as a CK5 interactor, which we confirmed by co-immunoprecipitation in several breast cancer models. We interrogated the dual functions of ß-catenin in relation to CK5. Knockout or knockdown of CK5 ablated ß-catenin transcriptional activity in response to progestins and Wnt stimuli. Conversely, CK5 induced by progestins or overexpression was sufficient to promote the loss of ß-catenin at the cell membrane and total E-cadherin loss. A breast cancer patient-derived xenograft showed similar loss of membrane ß-catenin and E-cadherin in CK5+ but not intratumoral CK5- cells and single-cell RNA sequencing found the top enriched pathways in the CK5+ cell cluster were cell junction remodeling and signaling. This report highlights that CK5 actively remodels cell morphology and that blockade of CK5-ß-catenin interaction may reverse the detrimental properties of CK5+ breast cancer cells.