Abstract: Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.

Abstract: Purpose: Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Experimental Design: Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results: ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions: ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. Clin Cancer Res; 22(5); 1130–7. ©2015 AACR. See related commentary by Gu and Fuqua, p. 1034

Abstract: The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.

Abstract: Bone is the preferred site for metastatic spread in women with advanced breast cancer, and skeletal complications is associated with high morbidity and mortality. Nearly 70% of luminal-type breast cancer patients with metastases to bone experience skeletal complications due to enhanced osteoclastogenesis (osteoclast activation) that increases bone resorption, pain, pathological fractures, spinal cord compression, and hypercalcemia. The present study was designed to determine the effect of a cruciferous vegetable component (Sulforaphane; SFN) on breast cancer-induced osteoclastogenesis. Osteoclast differentiation in mouse bone marrow monocytes (BMM) was inhibited significantly upon the addition of 10% conditioned-media (CM) from SFN-treated breast cancer cells belonging to different subtypes, including MDA-MB-231, MCF-7, and SK-BR-3, in comparison with corresponding control. PCR-based gene expression profiling identified a common set of genes downregulated by SFN treatment compared to vehicle-treated control in MDA-MB-231, MCF-7, and SK-BR-3 cells, including osteoclastogenesis promoting transcription factors (RUNX2, NF-κB, and SOX-9) and certain soluble molecules (MMP9, TNFα, and Cathepsin K). Many of these gene expression changes were confirmed in MDA-MB-231, MCF-7, and SK-BR-3 cells by RT-PCR or western blotting. To determine the in vivo efficacy, SFN was administrated orally (1 mg per mouse; three times/week) to athymic mice intracardially injected with MDA-MB-231-Luc cells. SFN administration significantly inhibited the multiplicity of bone metastases and increased the bone volume relative to total volume. SFN-mediated prevention of osteoclastogenesis was associated with a significant decrease in TRAP-positive osteoclasts in bones and the levels of Cathepsin K, IL-8, and RANKL in serum. Altogether, this study demonstrates, for the first time to the best of our knowledge, that SFN is a potent inhibitor of breast cancer-induced osteoclastogenesis and bone resorption in vitro and in vivo. This study was partly funded by a pilot project grant from the UPMC Hillman Cancer (NCI grant P30 CA047904; Robert L. Ferris- Principal Investigator) and RO1 CA142604 and CA129347 awarded by the National Cancer Institute (Shivendra Singh- Principal Investigator). Citation Format: Subrata K. Pore, Joseph D. Latoche, Carolyn J. Anderson, Juraj Adamik, Deborah L. Galson, Kurt R. Weiss, Boeun Lee, Rebecca J. Watters, Prashant N. Kumta, Shivendra V. Singh. Sulforaphane is a novel inhibitor of breast cancer-induced osteolytic bone resorption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5070.

Abstract: DNA sequencing has identified a limited number of driver mutations in metastatic breast cancer beyond single base-pair mutations in the estrogen receptor (ESR1). However, our previous studies and others have observed that structural variants, such as ESR1 fusions, may also play a role. Therefore, we expanded upon these observations by performing a comprehensive and highly sensitive characterization of copy-number (CN) alterations in a large clinical cohort of metastatic specimens. NanoString DNA hybridization was utilized to measure CN gains, amplifications, and deletions of 67 genes in 108 breast cancer metastases, and in 26 cases, the patient-matched primary tumor. For ESR1, a copyshift algorithm was applied to identify CN imbalances at exon-specific resolution and queried large data sets ( & gt;15,000 tumors) that had previously undergone next-generation sequencing (NGS). Interestingly, a subset of ER+ tumors showed increased ESR1 CN (11/82, 13%); three had CN amplifications (4%) and eight had gains (10%). Increased ESR1 CN was enriched in metastatic specimens versus primary tumors, and this was orthogonally confirmed in a large NGS data set. ESR1-amplified tumors showed a site-specific enrichment for bone metastases and worse outcomes than nonamplified tumors. No ESR1 CN amplifications and only one gain was identified in ER− tumors. ESR1 copyshift was present in 5 of the 11 ESR1-amplified tumors. Other frequent amplifications included ERBB2, GRB7, and cell-cycle pathway members CCND1 and CDK4/6, which showed mutually exclusivity with deletions of CDKN2A, CDKN2B, and CDKN1B. Implications: Copy-number alterations of ESR1 and key CDK pathway genes are frequent in metastatic breast cancers, and their clinical relevance should be tested further.

Abstract: Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer–induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. In vitro osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of runt-related transcription factor 2 (RUNX2) and nuclear factor-κB and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of NFKB1. Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating in vivo suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer–induced osteolytic bone resorption in vitro and in vivo.

Abstract: In high-grade chondrosarcoma, 5-year survival is lower than 50%. Therefore, it is important that preclinical models that mimic the disease with the greatest possible fidelity are used to potentially develop new treatments. Accumulating evidence suggests that two-dimensional (2-D) cell culture may not accurately represent the tumor’s biology. It has been demonstrated in other cancers that three-dimensional (3-D) cancer cell spheroids may recapitulate tumor biology and response to treatment with greater fidelity than traditional 2-D techniques. To our knowledge, the formation of patient-derived chondrosarcoma spheroids has not been described. Questions/purposes (1) Can patient-derived chondrosarcoma spheroids be produced? (2) Do spheroids recapitulate human chondrosarcoma better than 2-D cultures, both morphologically and molecularly? (3) Can chondrosarcoma spheroids provide an accurate model to test novel treatments? Methods Experiments to test the feasibility of spheroid formation of chondrosarcoma cells were performed using HT-1080, an established chondrosarcoma cell line, and two patient-derived populations, TP19-S26 and TP19-S115. Cells were cultured in flasks, trypsinized, and seeded into 96-well ultra-low attachment plates with culture media. After spheroids formed, they were monitored daily by bright-field microscopy. Spheroids were fixed using paraformaldehyde and embedded in agarose. After dehydration with isopropanol, paraffin-embedded spheroids were sectioned, and slides were stained with hematoxylin and eosin. To compare differences and similarities in gene expression between 2-D and 3-D chondrosarcoma cultures and primary tumors, and to determine whether these spheroids recapitulated the biology of chondrosarcoma, RNA was extracted from 2-D cultures, spheroids, and tumors. Quantitative polymerase chain reaction was performed to detect chondrosarcoma markers of interest, including vascular endothelial growth factor alpha, hypoxia-inducible factor 1α, COL2A1, and COL10A1. To determine whether 2-D and 3-D cultures responded differently to novel chondrosarcoma treatments, we compared their sensitivities to disulfiram and copper chloride treatment. To test their sensitivity to disulfiram and copper chloride treatment, 10,000 cells were seeded into 96-well plates for 2-D culturing and 3000 cells in each well for 3-D culturing. After treating the cells with disulfiram and copper for 48 hours, we detected cell viability using quantitative presto-blue staining and measured via plate reader. Results Cell-line and patient-derived spheroids were cultured and monitored over 12 days. Qualitatively, we observed that HT-1080 demonstrated unlimited growth, while TP19-S26 and TP19-S115 contracted during culturing relative to their initial size. Hematoxylin and eosin staining of HT-1080 spheroids revealed that cell-cell attachments were more pronounced at the periphery of the spheroid structure than at the core, while the core was less dense. Spheroids derived from the intermediate-grade chondrosarcoma TP19-S26 were abundant in extracellular matrix, and spheroids derived from the dedifferentiated chondrosarcoma TP19-S115 had a higher cellularity and heterogeneity with spindle cells at the periphery. In the HT-1080 cells, differences in gene expression were appreciated with spheroids demonstrating greater expressions of VEGF-α (1.01 ± 0.16 versus 6.48 ± 0.55; p = 0.003), COL2A1 (1.00 ± 0.10 versus 7.46 ± 2.52; p 〈 0.001), and COL10A1 (1.01 ± 0.19 versus 22.53 ± 4.91; p 〈 0.001). Differences in gene expressions were also noted between primary tumors, spheroids, and 2-D cultures in the patient-derived samples TP19-S26 and TP19-S115. TP19-S26 is an intermediate-grade chondrosarcoma. With the numbers we had, we could not detect a difference in VEGF-α and HIF1α gene expression compared with the primary tumor. COL2A1 (1.00 ± 0.14 versus 1.76 ± 0.10 versus 335.66 ± 31.13) and COL10A1 (1.06 ± 0.378 versus 5.98 ± 0.45 versus 138.82 ± 23.4) expressions were both greater in the tumor (p (COL2A1) 〈 0.001; p (COL10A1) 〈 0.0001) and 3-D cultures (p (COL2A1) = 0.004; p (COL10A1) 〈 0.0001) compared with 2-D cultures. We could not demonstrate a difference in VEGF-α and HIF1α expressions in TP19-S115, a dedifferentiated chondrosarcoma, in the tumor compared with 2-D and 3-D cultures. COL2A1 (1.00 ± 0.02 versus 1.86 ± 0.18 versus 2.95 ± 0.56) and COL10A1 (1.00 ± 0.03 versus 5.52 ± 0.66 versus 3.79 ± 0.36) expressions were both greater in spheroids (p (COL2A1) = 0.003; p (COL10A1) 〈 0.0001) and tumors (p (COL2A1) 〈 0.001; p (COL10A1) 〈 0.0001) compared with 2-D cultures. Disulfiram-copper chloride treatment demonstrated high cytotoxicity in HT-1080 and SW-1353 chondrosarcoma cells grown in the 2-D monolayer, but 3-D spheroids were highly resistant to this treatment. Conclusion We provide preliminary findings that it is possible to generate 3-D spheroids from chondrosarcoma cell lines and two human chondrosarcomas (one dedifferentiated chondrosarcoma and one intermediate-grade chondrosarcoma). Chondrosarcoma spheroids derived from human tumors demonstrated morphology more reminiscent of primary tumors than cells grown in 2-D culture. Spheroids displayed similar expressions of cartilage markers as the primary tumor, and we observed a higher expression of collagen markers in the spheroids compared with cells grown in monolayer. Spheroids also demonstrated greater chemotherapy resistance than monolayer cells, but more patient-derived spheroids are needed to further conclude that 3-D cultures may mimic the chemoresistance that chondrosarcomas demonstrate clinically. Additional studies on patient-derived chondrosarcoma spheroids are warranted. Clinical Relevance Chondrosarcomas demonstrate resistance to chemotherapy and radiation, and we believe that if they can be replicated, models such as 3-D spheroids may provide a method to test novel treatments for human chondrosarcoma. Additional comprehensive genomic studies are required to compare 2-D and 3-D models with the primary tumor to determine the most effective way to study this disease in vitro.