Project description:Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is frequently associated with favorable outcomes, as ~90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that ILC patients receiving adjuvant endocrine therapy may not benefit from improved outcomes versus other breast cancer patients. Based on these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome in vitro via gene expression microarray analyses and ER ChIP-Seq. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. Response to endocrine therapy was also examined in ILC cell lines. We identified 915 genes that were uniquely E2-regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also regulated in vivo in HCI-013. We observed that MM134 were de novo tamoxifen resistant, and were induced to grow by 4-hydroxytamoxifen, as well as other anti-estrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required FGFR1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cells were hormone deprived by replacing growth medium with IMEM+10% charcoal stripped serum for 3 days. Following deprivation, cells were treated for 3 or 24 hours with 1nM E2 or vehicle (0.01% EtOH) in biological quadruplicate. Following treatment, cells were lysed and RNA was harvested using the Illustra RNAspin Mini kit (GE Health). cRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies), and cRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA). cRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility.

Project description:Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is frequently associated with favorable outcomes, as ~90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that ILC patients receiving adjuvant endocrine therapy may not benefit from improved outcomes versus other breast cancer patients. Based on these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome in vitro via gene expression microarray analyses and ER ChIP-Seq. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. Response to endocrine therapy was also examined in ILC cell lines. We identified 915 genes that were uniquely E2-regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also regulated in vivo in HCI-013. We observed that MM134 were de novo tamoxifen resistant, and were induced to grow by 4-hydroxytamoxifen, as well as other anti-estrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required FGFR1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cells were hormone deprived by replacing growth medium with IMEM+2% charcoal stripped serum for 3 days. Following deprivation, cells were treated for 3 or 24 hours with 1nM E2 or vehicle (0.01% EtOH) in biological quadruplicate. Following treatment, cells were lysed and RNA was harvested using the Illustra RNAspin Mini kit (GE Health). cRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies), and cRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA). cRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility.

Project description:Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is frequently associated with favorable outcomes, as ~90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that ILC patients receiving adjuvant endocrine therapy may not benefit from improved outcomes versus other breast cancer patients. Based on these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome in vitro via gene expression microarray analyses and ER ChIP-Seq. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. Response to endocrine therapy was also examined in ILC cell lines. We identified 915 genes that were uniquely E2-regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also regulated in vivo in HCI-013. We observed that MM134 were de novo tamoxifen resistant, and were induced to grow by 4-hydroxytamoxifen, as well as other anti-estrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required FGFR1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cells were hormone deprived by replacing growth medium with IMEM+10% charcoal stripped serum for 3 days. Following deprivation, cells were treated for 3 or 24 hours with 1nM E2 or vehicle (0.01% EtOH) in biological quadruplicate. Following treatment, cells were lysed and RNA was harvested using the Illustra RNAspin Mini kit (GE Health). cRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies), and cRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA). cRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility.

Project description:Invasive lobular carcinoma (ILC) of the breast typically presents with clinical biomarkers consistent with a favorable response to endocrine therapies, and over 90 % of ILC cases express the estrogen receptor (ER). However, a subset of ILC cases may be resistant to endocrine therapies, suggesting that ER biology is unique in ILC. Using ILC cell lines, we previously demonstrated that ER regulates a distinct gene expression program in ILC cells, and we hypothesized that these ER-driven pathways modulate the endocrine response in ILC. One potential novel pathway is via the Wnt ligand WNT4, a critical signaling molecule in mammary gland development regulated by the progesterone receptor.The ILC cell lines MDA-MB-134-VI, SUM44PE, and BCK4 were used to assess WNT4 gene expression and regulation, as well as the role of WNT4 in estrogen-regulated proliferation. To assess these mechanisms in the context of endocrine resistance, we developed novel ILC endocrine-resistant long-term estrogen-deprived (ILC-LTED) models. ILC and ILC-LTED cell lines were used to identify upstream regulators and downstream signaling effectors of WNT4 signaling.ILC cells co-opted WNT4 signaling by placing it under direct ER control. We observed that ER regulation of WNT4 correlated with use of an ER binding site at the WNT4 locus, specifically in ILC cells. Further, WNT4 was required for endocrine response in ILC cells, as WNT4 knockdown blocked estrogen-induced proliferation. ILC-LTED cells remained dependent on WNT4 for proliferation, by either maintaining ER function and WNT4 regulation or uncoupling WNT4 from ER and upregulating WNT4 expression. In the latter case, WNT4 expression was driven by activated nuclear factor kappa-B signaling in ILC-LTED cells. In ILC and ILC-LTED cells, WNT4 led to suppression of CDKN1A/p21, which is critical for ILC cell proliferation. CDKN1A knockdown partially reversed the effects of WNT4 knockdown.WNT4 drives a novel signaling pathway in ILC cells, with a critical role in estrogen-induced growth that may also mediate endocrine resistance. WNT4 signaling may represent a novel target to modulate endocrine response specifically for patients with ILC.

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Endocrine response in invasive lobular carcinoma is characterized by unique estrogen-mediated gene expression and de novo tamoxifen resistance

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