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Triple-negative breast cancer (TNBC) has a high propensity for organ-specific metastasis. However, the underlying mechanisms are not well understood. Here we show that the primary TNBC tumor-derived C-X-C motif chemokines 1/2/8 (CXCL1/2/8) stimulate lung-resident fibroblasts to produce the C-C motif chemokines 2/7 (CCL2/7), which, in turn, activate cholesterol synthesis in lung-colonizing TNBC cells and induce angiogenesis at lung metastatic sites. Inhibiting cholesterol synthesis in lung-colonizing breast tumor cells by pulmonary administration of simvastatin-carrying HER3-targeting nanoparticles reduces angiogenesis and growth of lung metastases in a syngeneic TNBC mouse model. Our findings reveal a novel, chemokine-regulated mechanism for the cholesterol synthesis pathway and a critical role of metastatic site-specific cholesterol synthesis in the pulmonary tropism of TNBC metastasis. The study has implications for the unresolved epidemiological observation that use of cholesterol-lowering drugs has no effect on breast cancer incidence but can unexpectedly reduce breast cancer mortality, suggesting interventions of cholesterol synthesis in lung metastases as an effective treatment to improve survival in individuals with TNBC.

Triple-negative breast cancer (TNBC) is associated with a high rate of early recurrence and distant metastasis, frequent development of therapeutic resistance, and a poor prognosis. There is a lack of targeted therapies for this aggressive subtype of breast cancer. Identifying novel effective treatment modalities for TNBC remains an urgent and unmet clinical need. In this study, we investigated the anti-cancer effect of triptonide, a natural compound derived from the traditional Chinese medicinal herb Tripterygium wilfordii Hook F, in TNBC. We found that triptonide inhibits human TNBC cell growth in vitro and growth of TNBC xenograft mammary tumors. It induces apoptosis and suppresses stem-like properties as indicated by reduced mammosphere formation and aldehyde dehydrogenase activity in TNBC cells. We show that triptonide downregulates multiple cancer stem cell-associated genes but upregulates SNAI1 gene expression. In support of SNAI1 induction as a negative feedback response to triptonide treatment, in vitro-derived triptonide-resistant HCC1806 cells display a markedly higher expression of SNAI1 compared with parental cells. Mechanistically, the increase of SNAI1 expression is mediated by the activation of JNK signaling, but not by ERK and AKT, two well-established SNAI1 regulators. Furthermore, knockdown of SNAI1 in the triptonide-resistant HCC1806 cells increases sensitivity to triptonide and reduces mammosphere formation. These results indicate that triptonide holds promise as a novel anti-tumor agent for TNBC treatment. Our study also reveals a SNAI1-associated feedback mechanism which may lead to acquired resistance to triptonide.

Proteolysis targeting chimeric molecule ARV 825 causes ubiquitination of bromodomains resulting in their efficient degradation by proteasome activity. Bromodomain degradation down-regulates MYC transcription contributing to growth inhibition of various human cancers. We examined the therapeutic potential of ARV 825 against multiple myeloma (MM) cells both in vitro and in vivo. In a dose-dependent manner, ARV 825 inhibited proliferation of 13 human MM cell lines and three fresh patient samples, and was associated with cell cycle arrest and apoptosis. ARV 825 rapidly and efficiently degraded BRD 2 and BRD 4. Sensitivity of MM cells to ARV 825 was positively correlated with cereblon levels. RNA sequencing analysis showed important genes such as CCR1, RGS, MYB and MYC were down-regulated by ARV 825. A total of 170 small molecule inhibitors were screened for synergy with ARV 825. Combination of ARV 825 with inhibitor of either dual PI3K/mTOR, CRM1, VEGFR, PDGFRα/b, FLT3, IGF-1R, protein kinase C, CBP-EP300 or JAK1/2 showed synergistic activity. Importantly, ARV 825 significantly inhibited the growth of MM xenografts and improved mice survival. Taken together, our results, in conjunction with recently published findings, provide a rationale for investigating the efficacy of ARV 825 for MM, use of cereblon as a biomarker for therapy of MM patients, and the combination of ARV 825 with small molecule inhibitors to improve the outcome of MM patients.

Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 4573 and MC180295) against human multiple myeloma cellsin vitro.Short-hairpin RNA silencing of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation. Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single agent alone. Combination treatment synergistically inhibited multiple myeloma cells bothin vitroandin vivowith insignificant weight loss. The combination also resulted in marked increase of apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) is effective against MM cells.

Distant metastasis accounts for the vast majority of deaths in patients with cancer. Breast cancer exhibits a distinct metastatic pattern commonly involving bone, liver, lung, and brain. Breast cancer can be divided into different subtypes based on gene expression profiles, and different breast cancer subtypes show preference to distinct organ sites of metastasis. Luminal breast tumors tend to metastasize to bone while basal-like breast cancer (BLBC) displays a lung tropism of metastasis. However, the mechanisms underlying this organ-specific pattern of metastasis still remain to be elucidated. In this review, we will summarize the recent advances regarding the molecular signaling pathways as well as the therapeutic strategies for treating breast cancer lung metastasis.

The Forkhead box C1 (FOXC1) transcription factor is involved in normal embryonic development and regulates the development and function of many organs. Most recently, a large body of literature has shown that FOXC1 plays a critical role in tumor development and metastasis. Clinical studies have demonstrated that elevated FOXC1 expression is associated with poor prognosis in many cancer subtypes, such as basal-like breast cancer (BLBC). FOXC1 is highly and specifically expressed in BLBC as opposed to other breast cancer subtypes. Its functions in breast cancer have been extensively explored. This review will summarize current knowledge on the function and regulation of FOXC1 in tumor development and progression with a focus on BLBC, as well as the implications of these new findings in cancer diagnosis and treatment.

Summary Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres, referred to as mEBs, were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling pathways were hallmarks of 10-day differentiated mEBs. We then generated mammary-like organoids from 10-day mEBs using 3D floating mixed gel culture and a three-stage differentiation procedure. These organoids expressed common breast tissue, luminal, and basal markers, including estrogen receptor, and could be induced to produce milk protein. These results demonstrate that human iPSCs can be directed in vitro toward mammary lineage differentiation. Our findings provide an iPSC-based model for studying regulation of normal mammary cell fate and function as well as breast disease development., Graphical Abstract, Highlights • Enrichment of non-neural ectoderm-cell-containing spheres from human iPSCs • Generation of milk protein-expressing mammary-like organoids from human iPSCs • Identification of luminal- and myoepithelial-like cells in mammary-like organoids, In this article, Cui and colleagues show that human induced pluripotent stem cells can be induced to differentiate into mammary-like tissue structure in defined 3D culture conditions. These mammary-like tissues “in dish” comprise cells expressing biomarkers and phenotypes, such as milk secretion, associated with normal human breast tissue. Their study provides a model for dissecting human breast development and tumorigenesis.

Breast cancers arising in the setting of the hereditary breast cancer genes BRCA1 and BRCA2 are most commonly classified as basal-like breast cancer (BLBC) or luminal breast cancer, respectively. BLBC is an aggressive subtype of breast cancer associated with liver and lung metastases and poorer prognosis than other subtypes and for which chemotherapy is the only systemic therapy. Multiple immunohistochemical markers are used to identify the basal-like subtype, including the absence of estrogen receptor alpha, progesterone receptor, and human epidermal growth factor receptor 2. Forkhead box C1 (FOXC1) has been identified as a specific marker expressed in BLBC in general breast cancer cohorts. We examined an institutional cohort of breast cancer patients with germline BRCA1 (n=46) and BRCA2 (n=35) mutations and found that FOXC1 expression on immunohistochemical staining is associated with BRCA1 vs BRCA2 mutations [30/46 vs. 6/35]. In BRCA1 mutant tumors, FOXC1 was expressed in 28/31 BLBC tumors and 2/13 non-BLBC tumors, In BRCA2 mutant tumors, FOXC1 was expressed in 5/5 BLBC tumors and 1/30 non-BLBC tumors. In cell culture models of BRCA1-mutant breast cancer, FOXC1 is associated with increased proliferation and may serve as a marker for sensitivity to PARP-inhibitor therapy with olaparib.

Estrogen receptor-α (ERα) mediates the essential biological function of estrogen in breast development and tumorigenesis. Multiple mechanisms, including pioneer factors, coregulators and epigenetic modifications have been identified as regulators of ERα signaling in breast cancer. However, previous studies of ERα regulation have focused on luminal and HER2-positive subtypes rather than basal-like breast cancer (BLBC), in which ERα is underexpressed. In addition, mechanisms that account for the decrease or loss of ER expression in recurrent tumors after endocrine therapy remain elusive. Here, we demonstrate a novel FOXC1-driven mechanism that suppresses ERα expression in breast cancer. We find that FOXC1 competes with GATA-binding protein 3 (GATA3) for the same binding regions in the cis-regulatory elements upstream of the ERα gene and thereby downregulates ERα expression and consequently its transcriptional activity. The forkhead domain of FOXC1 is essential for the competition with GATA3 for DNA binding. Counteracting the action of GATA3 at the ERα promoter region, overexpression of FOXC1 hinders recruitment of RNA polymerase II and increases histone H3K9 trimethylation at ERα promoters. Importantly, ectopic FOXC1 expression in luminal breast cancer cells reduces sensitivity to estrogen and tamoxifen. Furthermore, in breast cancer patients with ER-positive primary tumors who received adjuvant tamoxifen treatment, FOXC1 expression is associated with decreased or undetectable ER expression in recurrent tumors. Our findings highlight a clinically relevant mechanism that contributes to the low or absent ERα expression in BLBC. This study suggests a new paradigm to study ERα regulation during breast cancer progression and indicates a role of FOXC1 in the modulation of cellular response to endocrine treatment.

Resistance to anti-tumor therapeutics is an important clinical problem. Tumor-targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a "Trojan-Horse" approach to combating these tumors by using a receptor-targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non-covalent self-assembly with therapeutic cargo, forming HER3-homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re-introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here.

Background The pathogenesis of human basal-like breast cancer (BLBC) is not well understood and patients with BLBC have a poor prognosis. Expression of the epidermal growth factor receptor (EGFR) and nuclear factor-κB (NF-κB) is well-known to be upregulated in BLBC. The forkhead box C1 (FOXC1) transcription factor, an important prognostic biomarker specific for BLBC, has been shown to be induced by EGF and is critical for EGF effects in breast cancer cells. How FOXC1 is transcriptionally activated in BLBC is not clear. Methods Luciferase reporter assays were performed to show that NF-κB-p65 enhances FOXC1 promoter activity in BLBC cells (MDA-MB-468). Electrophoretic mobility shift assay, biotinylated oligonucleotide precipitation assay, and chromatin immunoprecipitation assay were used to show that NF-κB interacts and binds to the promoter region of FOXC1. Results In this study, we demonstrate that NF-κB is a pivotal mediator of the EGF/EGFR regulation of FOXC1 expression by binding to the FOXC1 promoter to activate FOXC1 transcription. Loss or inhibition of NF-κB diminished FOXC1 expression. Conclusion Collectively, our findings reveal a novel EGFR-NF-κB-FOXC1 signaling axis that is critical for BLBC cell function, supporting the notion that intervention in the FOXC1 pathway may provide potential modalities for BLBC treatment. Electronic supplementary material The online version of this article (doi:10.1186/s12964-017-0180-3) contains supplementary material, which is available to authorized users.

Triple-negative breast cancer (TNBC) has high rates of local recurrence and distant metastasis, partially due to its high invasiveness. The Forkhead box C1 (FOXC1) transcription factor has been shown to be specifically overexpressed in TNBC and associated with poor clinical outcome. How TNBC's high invasiveness is driven by FOXC1 and its downstream targets remains poorly understood. In the present study, pathway-specific PCR array assays revealed that WNT5A and matrix metalloproteinase-7 (MMP7) were upregulated by FOXC1 in TNBC cells. Interestingly, WNT5A mediates the upregulation of MMP7 by FOXC1 and the WNT5A-MMP7 axis is essential for FOXC1-induced invasiveness of TNBC cells in vitro. Xenograft models showed that the lung extravasation and metastasis of FOXC1-overexpressing TNBC cells were attenuated by knocking out WNT5A, but could be restored by MMP7 overexpression. Mechanistically, FOXC1 can bind directly to the WNT5A promoter region to activate its expression. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), coupled with mass spectrometry, identified FOXC1-interacting proteins including a group of heterogeneous nuclear ribonucleoproteins involved in WNT5A transcription induction. Finally, we found that WNT5A activates NF-κB signaling to induce MMP7 expression. Collectively, these data demonstrate a FOXC1-elicited non-canonical WNT5A signaling mechanism comprising NF-κB and MMP7 that is essential for TNBC cell invasiveness, thereby providing implications toward developing an effective therapy for TNBC.

Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFβ-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis and development of regenerative medicine approaches.

BACKGROUND Diagnosis of basal-like breast cancer (BLBC) remains a bottleneck to conducting effective clinical trials for this aggressive subtype. We postulated that elevated expression of Forkhead Box transcription factor C1 (FOXC1) is a simple and accurate diagnostic biomarker for BLBC. METHODS Accuracy of FOXC1 expression in identifying BLBC was compared with the PAM50 gene expression panel in gene expression microarray (GEM) (n = 1992) and quantitative real-time polymerase chain reaction (qRT-PCR) (n = 349) datasets. A FOXC1-based immunohistochemical (IHC) assay was developed and assessed in 96 archival formalin-fixed, paraffin-embedded (FFPE) breast cancer samples that also underwent PAM50 profiling. All statistical tests were two-sided. RESULTS A FOXC1-based two-tier assay (IHC +/- qRT-PCR) accurately identified BLBC (AUC = 0.88) in an independent cohort of FFPE samples, validating the accuracy of FOXC1-defined BLBC in GEM (AUC = 0.90) and qRT-PCR (AUC = 0.88) studies, when compared with platform-specific PAM50-defined BLBC. The hazard ratio (HR) for disease-specific survival in patients having FOXC1-defined BLBC was 1.71 (95% CI = 1.31 to 2.23, P < .001), comparable to PAM50 assay-defined BLBC (HR = 1.74, 95% CI = 1.40 to 2.17, P < .001). FOXC1 expression also predicted the development of brain metastasis. Importantly, unlike triple-negative or Core Basal IHC definitions, a FOXC1-based definition is able to identify BLBC in both ER+ and HER2+ patients. CONCLUSION A FOXC1-based two-tier assay, by virtue of being rapid, simple, accurate, and cost-effective may emerge as the diagnostic assay of choice for BLBC. Such a test could substantially improve clinical trial enrichment of BLBC patients and accelerate the identification of effective chemotherapeutic options for this aggressive disease.

Background We investigated the effect of basal protein expression on trastuzamab response in patients with HER2-positive (HER2 + ) breast cancer who received trastuzamab (T) and in HER2 + breast cancer cell lines. Patients and Methods Expression of cytokeratin (CK) 5/6, CK14, and epidermal growth factor receptor (EGFR) was evaluated after immunohistochemical staining in paraffin-embedded tissue of 97 patients with stage I to III HER2 + breast cancer treated with chemotherapy/T. Groups with and without basal protein expression were compared with respect to clinicopathologic parameters and survival. We treated 4 cell lines (2 basal-HER2 [HCC1569, HCC1954] and 2 nonbasal HER2 [BT474, SKBR3]) each with vehicle, T 20 μg/mL, paclitaxel 0.01 μM (P), and T with P (T + P). Cell viability was assessed and HER2 pathway suppression was compared between groups using immunoblot analysis. Mammosphere formation was used to assess breast cancer stem cell properties. Results EGFR expression was significantly associated with cancer-specific survival (CSS) ( P = .05). CK5/6 expression strongly correlated with overall and disease-free survival, and CSS ( P = .03, P = .04, and P = .03, respectively). Statistical significance was maintained for EGFR and CK5/6 after adjustment for covariates. CK14 was not associated with survival. All cell lines expressed similar levels of HER2. T and P alone inhibited proliferation of nonbasal cell lines; T + P had an additive cytotoxic effect. Basal cells were resistant to T, P inhibited proliferation, but T + P had no additive cytotoxic effect on cell growth in basal cells. Immunoblot analysis showed a significant decrease in phosphorylated Akt levels after treatment with T or T + P in nonbasal cells but not in basal cells. Akt blockade suppressed growth of basal and nonbasal HER2 + cells. Furthermore, basal HER2 cell lines had increased mammosphere formation, which suggests increased stem cell properties compared with nonbasal HER2 cell lines. Conclusion CK5/6 and EGFR expression are predictive of worse prognosis in HER2 + breast cancer patients treated with T. Basal HER2 breast cancer cell lines are resistant to trastuzamab, which is mediated through the Akt pathway; AKT inhibition abrogates this resistance. Basal HER2 cell lines also have increased stem cell properties, which might play a role in the resistance pathway.

Vitamin A (VA) is required for normal fetal development and successful pregnancy. Excessive VA intake during pregnancy may lead to adverse maternal and fetal effects. Cytochrome P450 26A1 (cyp26a1), a retinoic acid (RA)-metabolizing enzyme, is involved in VA metabolism. It has been shown that cyp26a1 is expressed in female reproductive tract, especially in uterus. In order to investigate the role of cyp26a1 during pregnancy, we constructed a recombinant plasmid DNA vaccine encoding cyp26a1 protein and immunized mice with the plasmid. Compared to control groups, the pregnancy rate of the cyp26a1 plasmid-immunized mice were significantly decreased (P

SummaryThe mesoderm- and epithelial-mesenchymal transition-associated transcription factor FOXC1 is specifically overexpressed in basal-like breast cancer (BLBC), but its biochemical function is not understood. Here, we demonstrate that FOXC1 controls cancer stem cell (CSC) properties enriched in BLBC cells via activation of Smoothened (SMO)-independent Hedgehog (Hh) signaling. This non-canonical activation of Hh is specifically mediated by Gli2. Furthermore, we show that the N-terminal domain of FOXC1 (aa 1–68) binds directly to an internal region (aa 898–1168) of Gli2, enhancing the DNA-binding and transcription-activating capacity of Gli2. FOXC1 expression correlates with that of Gli2 and its targets in human breast cancers. Moreover, FOXC1 overexpression reduces sensitivity to anti-Hedgehog (Hh) inhibitors in BLBC cells and xenograft tumors. Together, these findings reveal FOXC1-mediated non-canonical Hh signaling that determines the BLBC stem-like phenotype and anti-Hh sensitivity, supporting inhibition of FOXC1 pathways as potential approaches for improving BLBC treatment.