Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

Progesterone downregulation of miR-141 contributes to expansion of stem-like breast cancer cells through maintenance of progesterone receptor and Stat5a.

Progesterone (P4) has emerged as an important hormone-regulating mammary stem cell (MaSC) populations. In breast cancer, P4 and synthetic analogs increase the number of stem-like cells within luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancers. These cells gain expression of de-differentiated cell markers CD44 and cytokeratin 5 (CK5), lose luminal markers ER and PR, and are more therapy resistant. We previously described that P4 downregulation of microRNA (miR)-29a contributes to the expansion of CD44(high) and CK5(+) cells. Here we investigated P4 downregulation of miR-141, a member of the miR-200 family of tumor suppressors, in facilitating an increase in stem-like breast cancer cells. miR-141 was the sole member of the miR-200 family P4-downregulated at the mature miRNA level in luminal breast cancer cell lines. Stable inhibition of miR-141 alone increased the CD44(high) population, and potentiated P4-mediated increases in both CD44(high) and CK5(+) cells. Loss of miR-141 enhanced both mammosphere formation and tumor initiation. miR-141 directly targeted both PR and signal transducer and activator of transcription 5A (Stat5a), transcription factors important for MaSC expansion. miR-141 depletion increased PR protein levels, even in cell lines where PR expression is estrogen dependent. Stat5a suppression via small interfering RNA or a small-molecule inhibitor reduced the P4-dependent increase in CK5(+) and CD44(high) cells. These data support a mechanism by which P4-triggered loss of miR-141 facilitates breast cancer cell de-differentiation through deregulation of PR and Stat5a, two transcription factors important for controlling mammary cell fate.

Progestin suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4.

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER)- and progesterone receptor (PR)-positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5) and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44(+) cell population. Downregulation of miR-29 members potentiates the expansion of CK5(+) and CD44(+) cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism, whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone-responsive breast cancers.

Exogenous Bcl-xL fusion protein spares neurons after spinal cord injury.

Spinal cord injury (SCI) induces neuronal death, including apoptosis, which is completed within 24 hr at and around the impact site. We identified early proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-xL, Bcl-2, and Bcl-w, using Affymetrix DNA microarrays. Because Bcl-xL is the most robustly expressed antiapoptotic Bcl-2 molecule in adult central nervous system, we decided to characterize better the effect of SCI on Bcl-xL expression. We found Bcl-xL expressed robustly throughout uninjured spinal cord in both neurons and glia cells. We also found Bcl-xL localized in different cellular compartments: cytoplasmic, mitochondrial, and nuclear. Bcl-xL protein levels decreased in the cytoplasm and mitochondria 2 hr after SCI and persisted for 24 hr. To test the contribution of proapoptotic decreases in Bcl-xL to neuronal death, we augmented endogenous Bcl-xL levels by administering Bcl-xL fusion protein (Bcl-xL FP) into injured spinal cords. Bcl-xL FP significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death. Because Bcl-xL FP increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI.