TFEB and TFE3 Depletion Increases Cancer Stem Cell Characteristics and EMT in Triple-Negative Breast Cancer.

Breast cancer is currently the second leading cause of death for women worldwide. One subtype of breast cancer with poor prognosis is triple-negative breast cancer (TNBC), which is characterized by the lack of expression of estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. TNBC is difficult to treat due to its drug resistance and aggressiveness. Tumor invasiveness is driven by epithelial-mesenchymal transition (EMT), which allows cells to acquire mobility to metastasize. EMT is also relevant to maintaining cancer stem cell (CSC) characteristics. Drug resistance is driven by CSCs, which are slow to divide, thus surviving chemotherapies, the standard of treatment. Thus, new treatments that inhibit CSCs and/or EMT in TNBC are highly desirable. TFEB and TFE3, two transcription factors of the MiT-TFE family that operate as a complex, are involved in regulating lysosome biogenesis and autophagy. Our preliminary data and other studies have shown that TFEB and TFE3 regulate pluripotency and EMT in various in vitro models such as gastric and pancreatic cancer, but this regulation is poorly understood in TNBC. To develop new targeted therapies for TNBC, this study aimed to investigate the effects of depleting TFEB and TFE3 on CSC abundance and EMT. CSC renewal capacity was assessed via mammosphere assays and flow cytometry using CD44 and ALDH1A. The MDA MB 231 TFEBKO cells grew the highest number and largest mammospheres compared to the control, TFE3 knock-down, and double knock-out cell lines. TFEBKO and TFEBKOTFE3sh showed increases in CD44 expression and ALDH1A activity. While ALDH1A positivity was 1.16% in controls, TFEBKO and TFEBKOTFE3sh significantly increased the population to 22.6% and 21.9%, respectively. Western blotting and a wound healing assay were used to evaluate EMT marker expression and migration, respectively. Quantification revealed that TFEBKO had a two-fold increase in protein expression levels of EMT markers Vimentin, Snail, and Slug, while TFE3sh showed higher protein expression levels of Vimentin and Slug. TFEBKOTFE3sh showed higher protein expression levels of Slug. The TFEBKO cell line had the highest rate of migration when compared to the control. In conclusion, the data showed that knockout of TFEB and combined knockout of TFEB and TFE3 resulted in an increase in stem cell character and EMT, which could correlate to increased tumor metastasis and poorer prognosis. These results are promising to investigate how depletion or inhibition of TFEB and TFE3 can affect TNBC tumor cell stem cell character and EMT.