Elaine T. Alarid

Estrogen receptor (ER)-positive breast cancer is characterized by late recurrences following initial treatment. The epithelial cell fate transcription factor Grainyhead-like protein 2 (GRHL2) is overexpressed in ER-positive breast cancers and is linked to poorer prognosis as compared to ER-negative breast cancers. To understand how GRHL2 contributes to progression, GRHL2 was overexpressed in ER-positive cells. We demonstrated that elevated GRHL2 imparts plasticity with stem cell- and dormancy-associated traits. RNA sequencing and immunocytochemistry revealed that high GRHL2 not only strengthens the epithelial identity but supports a hybrid epithelial to mesenchymal transition (EMT). Proliferation and tumor studies exhibited a decrease in growth and an upregulation of dormancy markers, such as NR2F1 and CDKN1B. Mammosphere assays and flow cytometry revealed enrichment of stem cell markers CD44 and ALDH1, and increased self-renewal capacity. Cistrome analyses revealed a change in transcription factor motifs near GRHL2 sites from developmental factors to those associated with disease progression. Together, these data support the idea that the plasticity and properties induced by elevated GRHL2 may provide a selective advantage to explain the association between GRHL2 and breast cancer progression.

The purpose of this study was to identify critical pathways promoting survival of tamoxifen-tolerant, estrogen receptor α positive (ER+) breast cancer cells, which contribute to therapy resistance and disease recurrence. Gene expression profiling and pathway analysis were performed in ER+ breast tumors of patients before and after neoadjuvant tamoxifen treatment and demonstrated activation of the NF-κB pathway and an enrichment of epithelial-to mesenchymal transition (EMT)/stemness features. Exposure of ER+ breast cancer cell lines to tamoxifen, in vitro and in vivo, gives rise to a tamoxifen-tolerant population with similar NF-κB activity and EMT/stemness characteristics. Small-molecule inhibitors and CRISPR/Cas9 knockout were used to assess the role of the NF-κB pathway and demonstrated that survival of tamoxifen-tolerant cells requires NF-κB activity. Moreover, this pathway was essential for tumor recurrence following tamoxifen withdrawal. These findings establish that elevated NF-κB activity is observed in breast cancer cell lines under selective pressure with tamoxifen in vitro and in vivo, as well as in patient tumors treated with neoadjuvant tamoxifen therapy. This pathway is essential for survival and regrowth of tamoxifen-tolerant cells, and, as such, NF-κB inhibition offers a promising approach to prevent recurrence of ER+ tumors following tamoxifen exposure.Implications:Understanding initial changes that enable survival of tamoxifen-tolerant cells, as mediated by NF-κB pathway, may translate into therapeutic interventions to prevent resistance and relapse, which remain major causes of breast cancer lethality.

The purpose of this study was to identify critical pathways promoting survival of tamoxifen-tolerant, estrogen receptor α positive (ER+) breast cancer cells, which contribute to therapy resistance and disease recurrence. Gene expression profiling and pathway analysis were performed in ER+ breast tumors of patients before and after neoadjuvant tamoxifen treatment and demonstrated activation of the NF-κB pathway and an enrichment of epithelial-to mesenchymal transition (EMT)/stemness features. Exposure of ER+ breast cancer cell lines to tamoxifen, in vitro and in vivo, gives rise to a tamoxifen-tolerant population with similar NF-κB activity and EMT/stemness characteristics. Small-molecule inhibitors and CRISPR/Cas9 knockout were used to assess the role of the NF-κB pathway and demonstrated that survival of tamoxifen-tolerant cells requires NF-κB activity. Moreover, this pathway was essential for tumor recurrence following tamoxifen withdrawal. These findings establish that elevated NF-κB activity is observed in breast cancer cell lines under selective pressure with tamoxifen in vitro and in vivo, as well as in patient tumors treated with neoadjuvant tamoxifen therapy. This pathway is essential for survival and regrowth of tamoxifen-tolerant cells, and, as such, NF-κB inhibition offers a promising approach to prevent recurrence of ER+ tumors following tamoxifen exposure.Implications:Understanding initial changes that enable survival of tamoxifen-tolerant cells, as mediated by NF-κB pathway, may translate into therapeutic interventions to prevent resistance and relapse, which remain major causes of breast cancer lethality.

Supplemental Figure 1. Tamoxifen treatment induces expression of NFκBB-related genes. Supplementary Figure 2. Immune cell composition and numbers in the tumor microenvironment do not significantly change upon tamoxifen treatment. Supplementary Figure 3. NFκB pathway and EMT/stemness factors in tamoxifen-tolerant persisters in T47D cells. Supplementary Figure 4. Expression of p65 and p50 proteins in MCF-7 CRISPR/Cas9 knock out clones. Supplementary Figure 5. NFκB pathway is not directly activated by tamoxifen. Supplementary Figure 6. Sorted NFκB+ vs NFκBâ^' or unsorted tamoxifen-tolerant cells based on GFP expression were reseeded for another round of clonogenic assay and the %GFP+ population was quantified in the second round.

Supplemental Table 1. Patient characteristics.

Supplemental Table 3. GSEA enrichment plots

Supplemental Materials and Methods and Figure Legends

Supplemental Table 1. Patient characteristics.

Supplemental Table 3. GSEA enrichment plots

Supplemental Materials and Methods and Figure Legends