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Certain immune cells and inflammatory cytokines are essential components in the tumor microenvironment to promote breast cancer progression. To identify key immune players in the tumor microenvironment, we applied highly invasive MDA-MB-231 breast cancer cell lines to co-culture with human monocyte THP-1 cells and identified CXCL7 by cytokine array as one of the increasingly secreted cytokines by THP-1 cells. Further investigations indicated that upon co-culturing, breast cancer cells secreted CSF1 to induce expression and release of CXCL7 from monocytes, which in turn acted on cancer cells to promote FAK activation, MMP13 expression, migration, and invasion. In a xenograft mouse model, administration of CXCL7 antibodies significantly reduced abundance of M2 macrophages in tumor microenvironment, as well as decreased tumor growth and distant metastasis. Clinical investigation further suggested that high CXCL7 expression is correlated with breast cancer progression and poor overall survival of patients. Overall, our study unveils an important immune cytokine, CXCL7, which is secreted by tumor infiltrating monocytes, to stimulate cancer cell migration, invasion, and metastasis, contributing to the promotion of breast cancer progression.

The remarkable advances in next-generation sequencing technology have enabled the wide usage of sequencing as a clinical tool. To promote the advance of precision oncology for breast cancer in China, here we report a large-scale prospective clinical sequencing program using the Fudan-BC panel, and comprehensively analyze the clinical and genomic characteristics of Chinese breast cancer. The mutational landscape of 1,134 breast cancers reveals that the most significant differences between Chinese and Western patients occurred in the hormone receptor positive, human epidermal growth factor receptor 2 negative breast cancer subtype. Mutations in p53 and Hippo signaling pathways are more prevalent, and 2 mutually exclusive and 9 co-occurring patterns exist among 9 oncogenic pathways in our cohort. Further preclinical investigation partially suggests that NF2 loss-of-function mutations can be sensitive to a Hippo-targeted strategy. We establish a public database (Fudan Portal) and a precision medicine knowledge base for data exchange and interpretation. Collectively, our study presents a leading approach to Chinese precision oncology treatment and reveals potentially actionable mutations in breast cancer., Chinese breast cancer patients have not been well represented in clinical sequencing studies. Here the authors analyse the mutational landscape of 1,134 Chinese breast cancer patients, finding actionable targets and a higher prevalence of p53 and Hippo pathway mutations compared to Western cohorts.

Triple-negative breast cancer (TNBC) remains the most aggressive cluster of all breast cancers, which is due to its rapid progression, high probabilities of early recurrence, and distant metastasis resistant to standard treatment. Following the advances in cancer genomics and transcriptomics that can illustrate the comprehensive profiling of this heterogeneous disease, it is now possible to identify different subclasses of TNBC according to both intrinsic signals and extrinsic microenvironment, which have a huge influence on predicting response to established therapies and picking up novel therapeutic targets for each cluster. In this review, we summarize basic characteristics and critical subtyping systems of TNBC, and particularly discuss newly found prospective targets and relevant medications, which were proved promising in clinical trials, thus shedding light on the future development of precision treatment strategies.

Metastatic disease is responsible for over 90% of death in patients with breast cancer. Therefore, identifying the molecular mechanisms that regulate metastasis and developing useful therapies are crucial tasks. Long non-coding RNAs (lncRNAs), which are non-coding transcripts with >200 nucleotides, have recently been identified as critical molecules for monitoring cancer progression. This study examined the novel lncRNAs involved in the regulation of tumor progression in breast cancer. This study identified 73 metastasis-related lncRNA candidates from comparison of paired isogenic high and low human metastatic breast cancer cell lines, and their expression levels were verified in clinical tumor samples by using The Cancer Genome Atlas. Among the cell lines, a novel lncRNA, LOC550643, was highly expressed in breast cancer cells. Furthermore, the high expression of LOC550643 was significantly correlated with the poor prognosis of breast cancer patients, especially those with triple-negative breast cancer. Knockdown of LOC550643 inhibited cell proliferation of breast cancer cells by blocking cell cycle progression at S phase. LOC550643 promoted important in vitro metastatic traits such as cell migration and invasion. Furthermore, LOC550643 could inhibit miR-125b-2-3p expression to promote breast cancer cell growth and invasiveness. In addition, by using a xenograft mouse model, we demonstrated that depletion of LOC550643 suppressed the lung metastatic potential of breast cancer cells. Overall, our study shows that LOC550643 plays an important role in breast cancer cell metastasis and growth, and LOC550643 could be a potential diagnosis biomarker and therapeutic target for breast cancer.

TP53 is the most frequently mutated or deleted gene in triple negative breast cancer (TNBC). Both the loss of TP53 and the lack of targeted therapy are significantly correlated with poor clinical outcomes, making TNBC the only type of breast cancer that has no approved targeted therapies. Through in silico analysis, we identified POLR2A in the TP53-neighboring region as a collateral vulnerability target in TNBC tumours, suggesting that its inhibition via small interfering RNA (siRNA) may be an amenable approach for TNBC targeted treatment. To enhance bioavailability and improve endo/lysosomal escape of siRNA, we designed pH-activated nanoparticles for augmented cytosolic delivery of POLR2A siRNA (siPol2). Suppression of POLR2A expression with the siPol2-laden nanoparticles (siPol2@NPs) leads to enhanced growth reduction of tumours characterised by hemizygous POLR2A loss. These results demonstrate the potential of the pH-responsive nanoparticle and the precise POLR2A targeted therapy in TNBC harbouring the common TP53 genomic alteration.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with unfavorable prognosis and 5-year survival. The purpose of this study was to investigate the underlying mechanisms involved in TNBC progression. We determined that CD24 expression was elevated in highly lung and lymph node metastatic TNBC cells. CD24 depletion inhibited primary tumor growth and lymph node and lung metastasis and reduced the number of blood and lymphatic vessels in the tumor microenvironment. CD24 knockdown impaired EGFR/Met-mediated signaling and reduced lymphangiogenesis- and angiogenesis-related molecules, including vascular endothelial growth factors A and C, by promoting EGFR and Met protein instability via the lysosomal degradation pathway. CD24 monoclonal antibody treatment reduced lung metastasis and prolonged the survival in a lung metastasis mouse model. Clinical analyses revealed that the CD24high/METhigh “double-positive” signature identified a subset of TNBC patients with worst outcomes. We conclude that CD24 could be a therapeutic target by itself and in combination with the Met expression could be a good prognostic biomarker for TNBC patients.

Background. The CXC chemokines belong to a unique family of cytokines that participates in the progression and development of many malignant tumors. Evidence for the relationship between chemokine (C-X-C motif) receptor 2 (CXCR2) C1208T polymorphism and susceptibility to cancer remains inconsistent. Methods. Odds ratios (ORs), 95% confidence intervals (CIs), and combined analysis were used to investigate the effect of CXCR2 variation on cancer risk. Gene Set Enrichment Analysis (GSEA) and enzyme-linked immunosorbent assay (ELISA) were also used to evaluate the expression of CXCR2 in prostate cancer (PCA). Results. Across 11 case-control studies, 4,909 cases and 5,884 controls were involved in the current analysis. Individuals with a TT genotype were associated with increased risk of digestive cancer, compared to those with a TC+CC genotype (OR=1.16,95%CI=1.02-1.31,P=0.025). Individuals carrying the TT genotype had a 39% higher risk of urinary cancer than those carrying CC genotype (OR=1.39,95%CI=1.04-1.87,P=0.025). Individuals with a TT genotype showed a 56% augmented breast cancer risk, compared to those with a CC genotype (OR=1.56,95%CI=1.03-2.35,P=0.034). It was found that CXCR2 expression was downregulated in PCA. Compared with PCA subjects carrying the CC genotype, the expression of CXCR2 was decreased in patients with the TT genotype. Conclusions. The CXCR2 C1208T variation was associated with elevated risk of urinary, breast, and digestive cancer. However, the C1208T polymorphism was correlated with attenuated risk of lung cancer.

Tumor metastasis is the main reason for the high mortality rate of breast cancer patients and lymph node metastasis is the primary means of tumor spreading. Liposomes loaded with bufalin (BF), a steroidal inhibitor of various inflammatory signaling pathways in cancer, and superparamagnetic Fe3O4 nanoparticles were developed to inhibit lymphatic metastasis in breast cancer. Fe3O4 functioned as both a magnetic targeting and photothermal agent. In vivo studies using a mouse model of lymph metastatic breast cancer demonstrated that BF-Fe3O4-L(liposomes), administered by iv injection, accumulated in sentinel lymph nodes (SLNs) with high efficiency under magnetic direction and exhibited significant photothermal character under near-infrared (NIR) light irradiation. The combination of targeted delivery, photothermal and chemotherapeutic properties resulted in a 91% tumor inhibition rate in SLNs compared with untreated (PBS) controls and 81% lower incidence of pulmonary metastases formation. These findings reveal the significant potential of BF- Fe3O4-Ls for delivering -targeted, chemo-photothermal therapy in the treatment of metastasic cancer.

Migration and invasion are key properties of metastatic cancer cells. These properties can be acquired through intrinsic reprogramming processes such as epithelial-mesenchymal transition. In this study, we discovered an alternative “migration-by-tethering” mechanism through which cancer cells gain the momentum to migrate by adhering to mesenchymal stem cells or osteoblasts. This tethering is mediated by both heterotypic adherens junctions and gap junctions, and leads to a unique cellular protrusion supported by cofilin-coated actin filaments. Inhibition of gap junctions or depletion of cofilin reduces migration-by-tethering. We observed evidence of these protrusions in bone segments harboring experimental and spontaneous bone metastasis in animal models. These data exemplify how cancer cells may acquire migratory ability without intrinsic reprogramming. Furthermore, given the important roles of osteogenic cells in early-stage bone colonization, our observations raise the possibility that migration-by-tethering may drive the relocation of disseminated tumor cells between different niches in the bone microenvironment.

Treatment of triple-negative breast cancer (TNBC) is a challenge to oncologists. Currently, the lack of effective therapy has fostered a major effort to discover new targets and therapeutics to combat this disease. The recepteur d’origine nantais (RON) receptor has been implicated in the pathogenesis of TNBC. Clinical studies have revealed that aberrant RON expression is crucial in regulating TNBC malignant phenotypes. Increased RON expression also has prognostic value for breast cancer progress. These features provide the rationale to target RON for TNBC treatment. In this review, we discuss the importance of RON in TNBC tumorigenesis and the development of anti-RON antibody–drug conjugates (ADCs) for clinical application. The findings from preclinical studies lay the foundation for clinical trials of this novel biotherapeutic for TNBC therapy.

Cancer stem cells (CSCs) are rare cancer cells that are postulated to be responsible for cancer relapse and metastasis. However, CSCs are difficult to isolate and poorly understood. Here, a bioinspired approach for label‐free isolation and culture of CSCs, by microencapsulating one cancer cell in the nanoliter‐scale hydrogel core of each prehatching embryo‐like core–shell microcapsule, is reported. Only a small percentage of the individually microencapsulated cancer cells can proliferate into a cell colony. Gene and protein expression analyses indicate high stemness of the cells in the colonies. Importantly, the colony cells are capable of cross‐tissue multilineage (e.g., endothelial, cardiac, neural, and osteogenic) differentiation, which is not observed for “CSCs” isolated using other contemporary approaches. Further studies demonstrate the colony cells are highly tumorigenic, metastatic, and drug resistant. These data show the colony cells obtained with the bioinspired one‐cell‐culture approach are truly CSCs. Significantly, multiple pathways are identified to upregulate in the CSCs and enrichment of genes related to the pathways is correlated with significantly decreased survival of breast cancer patients. Collectively, this study may provide a valuable method for isolating and culturing CSCs, to facilitate the understanding of cancer biology and etiology and the development of effective CSC‐targeted cancer therapies., A bioinspired technology is developed to isolate cancer stem cells (CSCs) by culturing one cell in the nanoliter‐scale hydrogel core of the prehatching embryo‐like core–shell microcapsules. The CSCs are capable of cardiac, neural, osteogenic, and endothelial differentiation, and are highly tumorigenic, metastatic, and drug resistant. The technology may be valuable for understanding cancer biology/etiology and developing CSC‐targeted effective cancer therapies.

Bone is the most frequently affected organ by metastases of breast cancer and prostate cancer. Our knowledge on bone metastasis is extremely limited due to the lack of potent and efficient experimental models. We developed the "Bone-In-Culture Array (BICA)" platform to model the bone colonization of cancer cells in ex vivo cultures. The use of the BICA platform will facilitate in-depth mechanistic studies and high-throughput screening of drugs for bone metastasis.

Purpose Triple-negative breast cancer (TNBC) is highly malignant and has poor prognosis and a high mortality rate. The lack of effective therapy has spurred our investigation of new targets for treating this malignant cancer. Here, we identified RON (macrophage-stimulating 1 receptor) and MET (MET proto-oncogene, receptor tyrosine kinase) as a prognostic biomarker and therapeutic targets for potential TNBC treatment.Materials and MethodsWe analyzed RON and MET expression in 187 primary TNBC clinical samples with immunohistochemistry. We validated the targeted therapeutic effects of RON and MET in TNBC using three tyrosine kinase inhibitors (TKIs): BMS-777607, INCB28060, and tivantinib. The preclinical therapeutic efficacy of the TKIs was mainly estimated using a TNBC xenograft model.ResultsPatients with TNBC had widespread, abnormal expression of RON and MET. There was RON overexpression, MET overexpression, and RON and MET co-overexpression in 63 (33.7%), 63 (33.7%), and 43 cases (23.0%), respectively, which had poor prognosis and short survival. In vivo, the TKI targeting RON ant MET inhibited the activation of the downstream signaling molecules, inhibited TNBC cell migration and proliferation, and increased TNBC cell apoptosis; in the xenograft model, they significantly inhibited tumor growth and shrank tumor volumes. The TKI targeting RON and Met, such as BMS-777607 and tivantinib, yielded stronger anti-tumor effects than INCB28060.ConclusionRON and MET co-overexpression can be significant pathological characteristics in TNBC for poor prognosis. TKIs targeting RON and MET have stronger drug development potential for treating TNBC.

Although metabolic reprogramming is recognized as a hallmark of tumorigenesis and progression, little is known about metabolic enzymes and oncometabolites that regulate breast cancer metastasis, and very few metabolic molecules have been identified as potential therapeutic targets. In this study, the transketolase (TKT) expression correlated with tumor size in the 4T1/BALB/c syngeneic model. In addition, TKT expression was higher in lymph node metastases compared with primary tumor or normal tissues of patients, and high TKT levels were associated with poor survival. Depletion of TKT or addition of alpha-ketoglutarate (αKG) enhanced the levels of tumor suppressors succinate dehydrogenase and fumarate hydratase (FH), decreasing oncometabolites succinate and fumarate, and further stabilizing HIF prolyl hydroxylase 2 (PHD2) and decreasing HIF1α, ultimately suppressing breast cancer metastasis. Reduced TKT or addition of αKG mediated a dynamic switch of glucose metabolism from glycolysis to oxidative phosphorylation. Various combinations of the TKT inhibitor oxythiamine, docetaxel, and doxorubicin enhanced cell death in triple-negative breast cancer (TNBC) cells. Furthermore, oxythiamine treatment led to increased levels of αKG in TNBC cells. Together, our study has identified a novel TKT-mediated αKG signaling pathway that regulates breast cancer oncogenesis and can be exploited as a modality for improving therapy. Significance: These findings uncover the clinical significance of TKT in breast cancer progression and metastasis and demonstrate effective therapy by inhibiting TKT or by adding αKG. Cancer Res; 78(11); 2799–812. ©2018 AACR.

The early onset breast cancer patients (age ≤ 40) often display higher incidence of axillary lymph node metastasis, and poorer five-year survival than the late-onset patients. To identify the genes and molecules associated with poor prognosis of early onset breast cancer, we examined gene expression profiles from paired breast normal/tumor tissues, and coupled with Gene Ontology and public data base analysis. Our data showed that the expression of GAS7b gene was lower in the early onset breast cancer patients as compared to the elder patients. We found that GAS7 was associated with CYFIP1 and WAVE2 complex to suppress breast cancer metastasis via blocking CYFIP1 and Rac1 protein interaction, actin polymerization, and β1-integrin/FAK/Src signaling. We further demonstrated that p53 directly regulated GAS7 gene expression, which was inversely correlated with p53 mutations in breast cancer specimens. Our study uncover a novel regulatory mechanism of p53 in early onset breast cancer progression through GAS7–CYFIP1-mediated signaling pathways.

Estrogen receptor-positive (ER+) breast cancer exhibits a strong bone tropism in metastasis. How the bone microenvironment (BME) impacts ER signaling and endocrine therapy remains poorly understood. Here, we discover that the osteogenic niche transiently and reversibly reduces ER expression and activities specifically in bone micrometastases (BMMs), leading to endocrine resistance. As BMMs progress, the ER reduction and endocrine resistance may partially recover in cancer cells away from the osteogenic niche, creating phenotypic heterogeneity in macrometastases. Using multiple approaches, including an evolving barcoding strategy, we demonstrated that this process is independent of clonal selection, and represents an EZH2-mediated epigenomic reprogramming. EZH2 drives ER+ BMMs toward a basal and stem-like state. EZH2 inhibition reverses endocrine resistance. These data exemplify how epigenomic adaptation to BME promotes phenotypic plasticity of metastatic seeds, fosters intra-metastatic heterogeneity, and alters therapeutic responses. Our study provides insights into the clinical enigma of ER+ metastatic recurrences despite endocrine therapies.

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

Glucocorticoids (GCs) are widely prescribed as adjuvant therapy for breast cancer patients. Unlike other steroid hormone receptors, the GC receptor is not considered an oncogene. Research in the past few years has revealed the complexity of GC-mediated signaling, but it remains puzzling whether GCs promote or inhibit tumor progression in different cancer types. Here we evaluated the potential of using a synthetic GC, dexamethasone (DEX), in the treatment of breast cancer. We found that the administration of low-dose DEX suppressed tumor growth and distant metastasis in the MCF-7 and MDA-MB-231 xenograft mouse model, whereas treatment with high-dose DEX enhanced tumor growth and metastasis, respectively. Treatment of breast cancer cells with DEX inhibited cell adhesion, migration, and invasion in a dose-dependent manner. The DEX-mediated inhibition of cell adhesion, migration, and invasion is partly through induction of microRNA-708 and subsequent Rap1B-mediated signaling in MDA-MB-231 cells. On the other hand, in MCF-7 cells, DEX-suppressed cell migration is independent from microRNA-708 mediated signaling. Overall, our data reveal that DEX acts as a double-edged sword during breast-cancer progression and metastasis: Lower concentrations inhibit breast cancer tumor growth and metastasis, whereas higher concentrations may play an undesired role to promote breast cancer progression.

MicroRNAs (miRNAs) function as genetic modulators that regulate gene expression and are involved in a wide range of biological roles, including tumor cell migration and invasion. In the present study, we demonstrated that the migration and invasion activity in MDA-MB-231 breast cancer cells could be directly influenced by altering miR-146a expression. The expression of RhoA and miR-146a in the breast cancer cells showed an inverse correlation. Upregulation of miR-146a in the MDA-MB‑231 breast cancer cells by transfection of miR-146a mimics resulted in decreased RhoA protein levels. Conversely, downregulation of miR-146a by transfection of miR-146a inhibitor resulted in increased RhoA protein levels. To confirm the fact that RhoA is a potential target of miR-146a, luciferase reporter containing the RhoA 3' untranslated region (3'UTR) was constructed. The results demonstrated that the luciferase reporter activity was reduced after overexpression of miR-146a. Moreover, the luciferase reporter which was constructed with the RhoA 3'UTR mutant did not show significantly altered luciferase reporter activity. Furthermore, after treatment with the RhoA inhibitor exoenzyme C3 transferase protein, the migratory capacity of the MDA-MB-231 cells was not significantly altered even though the amount of miR-146a was changed. Our results indicate that miR-146a functions as a tumor suppressor in breast cancer cells. Downregulation of the expression of miR-146a increased the migration of MDA-MB-231 cells, due to the upregulation of RhoA expression.

Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby sustaining rapid proliferation. Recently, new evidence has emerged that metabolic dysregulation may occur at early-stages of neoplasia and critically contribute to cancer initiation. Here, our bioinformatics analysis of microarray data from early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of canonical glycolytic genes, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human mammary epithelial cells (hMECs) up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation. Knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Mechanistically, 14-3-3ζ overexpression activates the MEK-ERK-CREB axis, which subsequently up-regulates LDHA. In vivo, inhibiting the activated the MEK/ERK pathway in 14-3-3ζ-overexpressing hMEC-derived MCF10DCIS.COM lesions led to effective inhibition of tumor growth. Therefore, targeting the MEK/ERK pathway could be an effective strategy for intervention of 14-3-3ζ-overexpressing early breast lesions. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis and transcriptionally up-regulating LDHA, thereby contributes to human breast cancer initiation.

TGFβ signaling inhibits cell proliferation to block cancer initiation, yet it also enhances metastasis to promote malignancy during breast cancer development. The mechanisms underlying these differential effects are still unclear. Here, we report that HER2/EGFR signaling switches TGFβ function in breast cancer cells from antiproliferation to cancer promotion. Inhibition of HER2/EGFR activity attenuated TGFβ–induced epithelial–mesenchymal transition and migration but enhanced the antiproliferative activity of TGFβ. Activation of HER2/EGFR induced phosphorylation of Smad3 at Ser208 of the linker region through AKT, which promoted the nuclear accumulation of Smad3 and subsequent expression of the genes related to EMT and cell migration. In contrast, HER2/EGFR signaling had no effects on the nuclear localization of Smad2. Knockdown of Smad3, but not Smad2, blocked TGFβ–induced breast cancer cell migration. We observed a positive correlation between the nuclear localization of Smad3 and HER2 activation in advanced human breast cancers. Our results demonstrate a key role for HER2/EGFR in differential regulation of Smad3 activity to shift TGFβ function from antitumorigenic to protumorigenic during breast cancer development. Significance: TGFβ signaling can shift from inhibiting to promoting breast cancer development via HER2/EGFR AKT-mediated phosphorylation of Smad3 at S208, enhancing its nuclear accumulation and upregulation of EMT-related genes. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6073/F1.large.jpg. Cancer Res; 78(21); 6073–85. ©2018 AACR.

SummaryBasal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

Breast cancer cell lines and mouse models are valuable tools for investigating the biology of and developing potential therapeutics for human breast carcinoma. The PTEN-/-/NIC mouse is a genetically engineered mouse model for ErbB2/Neu-overexpressing/‑PTEN deficient breast carcinoma with histopathological and molecular features relevant to the luminal subtype of primary human breast cancer. However, the PTEN-/-/NIC model develops multifocal and aggressive mammary tumors with a short life-span, which greatly impedes its preclinical usage. To complement the genetic engineering approach and to facilitate the future application of this model, in the present study, two newly established cell lines, NICP20 and NICP21, from PTEN-/-/NIC mammary tumors are described. These NICP20 and NICP21 cells retained the crucial molecular phenotype similar to the origin, as confirmed by genotyping and western blot analysis. These cells induced tumors in immunocompetent syngeneic mice by mammary fat pad injection and produced lung metastasis when injected intravenously. Tumors induced by these cells displayed luminal‑like histologic morphology and hyperactivation of Akt which are similar to PTEN-/-/NIC tumors. Immunohistochemical staining also revealed that tumors induced by the NICP20 and NICP21 cells showed a high proliferative level, comparable angiogenesis and T-cell infiltration properties similar to PTEN-/-/NIC tumors. Therefore, these NICP20 and NICP21 cells represent an alternative and useful model system to enhance our understanding of the nature of ErbB2-positive breast cancers, particularly accompanying PTEN loss and to facilitate further experimental therapeutic studies.

The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic metastases, are likely to differ from these tumours due to distinct cancer-microenvironment crosstalk in distant organs. Here, to recapitulate such differences, we established an ex vivo bone metastasis model, termed bone-in-culture array or BICA, by fragmenting mouse bones preloaded with breast cancer cells via intra-iliac artery injection. Cancer cells in BICA maintain features of in vivo bone micrometastases regarding the microenvironmental niche, gene expression profile, metastatic growth kinetics and therapeutic responses. Through a proof-of-principle drug screening using BICA, we found that danusertib, an inhibitor of the Aurora kinase family, preferentially inhibits bone micrometastases. In contrast, certain histone methyltransferase inhibitors stimulate metastatic outgrowth of indolent cancer cells, specifically in the bone. Thus, BICA can be used to investigate mechanisms involved in bone colonization and to rapidly test drug efficacies on bone micrometastases., The bone microenvironment may alter therapeutic responses of disseminated breast cancer cells. Here the authors establish an ex vivo bone metastasis model, termed BICA, to delineate the effects of bone microenvironment and to rapidly discover anti-metastasis drugs.

Metastasis is the predominant cause of death in breast cancer patients. Several lines of evidence have shown that microRNAs (miRs) can have an important role in cancer metastasis. Using isogenic pairs of low and high metastatic lines derived from a human breast cancer line, we have identified miR-149 to be a suppressor of breast cancer cell invasion and metastasis. We also identified GIT1 (G-protein-coupled receptor kinase-interacting protein 1) as a direct target of miR-149. Knockdown of GIT1 reduced migration/invasion and metastasis of highly invasive cells. Re-expression of GIT1 significantly rescued miR-149-mediated inhibition of cell migration/invasion and metastasis. Expression of miR-149 impaired fibronectin-induced focal adhesion formation and reduced phosphorylation of focal adhesion kinase and paxillin, which could be restored by re-expression of GIT1. Inhibition of GIT1 led to enhanced protein degradation of paxillin and α5β1 integrin via proteasome and lysosome pathways, respectively. Moreover, we found that GIT1 depletion in metastatic breast cancer cells greatly reduced α5β1-integrin-mediated cell adhesion to fibronectin and collagen. Low level of miR-149 and high level of GIT1 was significantly associated with advanced stages of breast cancer, as well as with lymph node metastasis. We conclude that miR-149 suppresses breast cancer cell migration/invasion and metastasis by targeting GIT1, suggesting potential applications of the miR-149-GIT1 pathway in clinical diagnosis and therapeutics.

Cancer biology and drug discovery are heavily dependent on conventional 2D cell culture systems. However, a 2D culture is significantly limited by its ability to reflect 'true biology' of tumor in vivo. Three-dimensional (3D) in vitro cell culture models have been introduced to aid cancer drug discovery by better modeling the tumor microenvironment. Here, decellularized lung scaffolds cultured with MCF-7 cancer cells were bioengineered as a platform to study tumor development and anti-cancer drug evaluation. Excellent cell compatibility of decellularized lung scaffolds promoted cell growth and proliferation. Multicellular tumor cell spheroids (tumoriods) were formed and enlarged exclusively in decellularized lung scaffolds over time. The expression of breast cancer biomarkers (BRCA1 and HER2) in MCF-7 cells significantly increased in the lung matrix compared to those cultured in 2D systems. Insufficient oxygen and nutrient diffusion into the internal surface of lung scaffolds resulted in intracellular hypoxia, quantified by a significant upregulation of HIF-1α protein expression compared to that of cell monolayers. Higher survival rates after exposure to 5-FU were observed in lung scaffolds (52.04%) compared to that in 2D systems (18.39%) on day 3 of culture. Overall, this new breast tumor system provides a promising platform to study breast cancer progression and develop new targeted therapeutic strategies.

Despite better control of early-stage disease and improved overall survival of patients with breast cancer, the incidence of life-threatening brain metastases continues to increase in some of these patients. Unfortunately, other than palliative treatments there is no effective therapy for this condition. In this study, we reveal a critical role for Src activation in promoting brain metastasis in a preclinical model of breast cancer and we show how Src-targeting combinatorial regimens can treat HER2+ brain metastases in this model. We found that Src was hyperactivated in brain-seeking breast cancer cells derived from human cell lines or from patients' brain metastases. Mechanistically, Src activation promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood–brain barrier. When combined with the EGFR/HER2 dual-targeting drug lapatinib, an Src-targeting combinatorial regimen prevented outgrowth of disseminated breast cancer cells through the induction of cell-cycle arrest. More importantly, this combinatorial regimen inhibited the outgrowth of established experimental brain metastases, prolonging the survival of metastases-bearing mice. Our results provide a rationale for clinical evaluation of Src-targeting regimens to treat patients with breast cancer suffering from brain metastasis. Cancer Res; 73(18); 5764–74. ©2013 AACR.

Aberrant expression of the RON receptor tyrosine kinase contributes to breast cancer malignancy. Although clinical trials of RON targeting are underway, the intriguing issue is the diversity of RON expression as evident by cancer cells expressing different variants including oncogenic RON160. The current study determines aberrant RON160 expression in breast cancer and its potential as a target for breast cancer therapy. Using mouse monoclonal antibody Zt/h12 in immunohistochemical staining of breast cancer tissue microarray, we observed that RON160 was expressed in high frequency in primary invasive ductal (77.2%, 61/79 cases), lobular (42.5%, 34/80 cases), and lymph node-involved (63.9%, 26/36 cases) breast cancer samples. Moreover, RON160 overexpression was predominantly observed in invasive ductal (26.6%, 21/79 cases) and lymph node-involved (33.3%, 12/36) cases. Among a panel of breast cancer cell lines analyzed, Du4475 cells naturally expressed RON160. Silencing RON160 expression by siRNA reduced Du4475 cell viability. Inhibition of RON160 signaling by tyrosine kinase inhibitor PHA665752 also suppressed Du4475 cell anchorage-independent growth and induced apoptotic cell death. Studies in vivo revealed that PHA665752 inhibited 3T3- RON160 and Du4475 cell-mediated tumor growth in mouse mammary fat pad. A 60% reduction in tumor volume compared to controls was achieved after a 13-day treatment. We conclude from these studies that RON160 is highly expressed in breast cancer and its signaling is integrated into cellular signaling network for tumor cell growth and survival. Experimental treatment by PHA665752 in Du4475 breast cancer xenograft model highlights the significance of RON160 as a drug target in molecular-targeted breast cancer therapy.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

Lipid-rich carcinoma of the breast is a rare variant of breast cancer, accounted for1% of all breast malignant tumors. We retrospectively analyzed the clinicopathologic characteristics of lipid-rich carcinomas of the breast. A panel of estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2 (HER-2), cytokeratin (CK) 5/6, CK14, and P63 was prepared for detection of lipid-rich carcinoma. Fluorescence in situ hybridization and electron microscope assays were performed for detecting HER-2 gene amplification and ultrastructure. Survival analysis were carried out using Kaplan-Meier and Cox regression methods. Receiver operating characteristic test was also performed. Estrogen receptor, CK5/6, CK14, and P63 were negative. Progesterone receptor (1/17) and HER-2 (17/17) were positive. HER-2 gene amplification was detected in all included cases (ratio values2.2). Ultrastructure showed fat droplet and electron-dense material in the cytoplasm. Statistical differences were detected among survival and age (P = .033), histologic grade (P = .004), lymph node involvement (P = .001), and HER-2 expression (P = .002), respectively, using Kaplan-Meier methods. Statistical differences were also detected (P = .017) using Cox regression methods. Receiver operating characteristic test displayed significant statistical differences; the prognosis had a correlation between HER-2 expression "+" and HER-2 expression "++" (P = .004). Lipid-rich carcinoma of the breast was an HER-2 overexpressing subtype of the breast carcinoma. Survival of lipid-rich carcinoma might be associated with age, histologic grade, lymph node involvement, and HER-2. The HER-2 expression, however, might play an important role in predicting the prognosis of lipid-rich carcinoma of the breast.

The objective of this study was to assess the anti-tumor efficacy of rapamycin alone or in combination with herceptin in breast cancer. A total of 20 human breast cancer lines were examined for expression of various receptor tyrosine kinases and activation of their down stream signaling molecules, as well as for their invasion and colony forming ability. The ErbB2 and PI3 kinase pathway inhibitors were tested for the inhibition on breast cancer cell growth and tumor development. Seven of the 20 lines displayed an elevated level of ErbB2, others had varying level of EGF, IGF-1 or insulin receptor. Over 30% of the lines also had constitutive activation of Akt and MAP kinase. The lines displayed a wide range of colony forming and invasion ability. The PI3 kinase pathway inhibitors LY294002 and rapamycin inhibited the colony forming ability of all of the lines with the ErbB2 overexpressing lines having a higher sensitivity. A similar trend was observed for inhibition of invasion by LY294002. Rapamycin alone and additively together with herceptin inhibited the breast cancer cell growth especially in ErbB2 overexpressing cells. Rapamycin and herceptin synergistically inhibited tumor growth and endpoint tumor load in a xenograft model using a MCF-7 subline and in a MMTV-ErbB2 transgenic model. Rapamycin and herceptin significantly reduced the level of cyclin D1 and D3 and increased the cleavage of caspase 3 suggesting an increased apoptosis. Our results suggest that rapamycin together with herceptin has an enhanced anti-cancer effect and could be developed as an improved therapeutic regimen for breast cancer. © 2007 Wiley-Liss, Inc.

Tumour necrosis factor-α (TNF-α) is critical in the regulation of inflammation and tumour progression. TNF-α-308G > A is associated with constitutively elevated TNF-α expression. The purpose of this study was to assess the association between TNF-α-308G > A and breast cancer (BC) risk by subtype and the connection between genotypes and clinical features of BC. A total of 768 patients and 565 controls were enrolled in this study and genotypes were detected using the TaqMan assay. No effect on susceptibility for any BC subtype was found for the TNF-α-308 polymorphism in our study or in the pooled meta-analysis. This polymorphism was shown to be associated with age at menarche in all BC and in progesterone receptor-negative BC. Interestingly, triple negative breast cancer (TNBC) patients with TNF-α-308A had an increased risk of distant tumour metastasis (OR = 3.80, 95% CI: 1.31–11.02, P = 0.009). Multi-regression analysis showed that TNF-α-308A was also a risk factor for distant tumour metastasis after adjustment for tumour size and lymph node metastasis status (OR= 6.26, 95% CI: 1.88–20.87, P = 0.003). These findings indicate that TNF-α might play a distinct role in the progression of TNBC, especially in distant tumour metastasis of TNBC.

We recently discovered that bone micrometastases of breast cancer predominantly reside in the microenvironment termed the “osteogenic niche”. The heterotypic adherens junctions between cancer cells and osteogenic cells promote early-stage bone colonization by activating the mTOR pathway in cancer cells. Here, we discuss a few questions raised by these findings.

A requirement for Mouse Double Minute 2 (MDM2) oncogene activation has been suggested to be associated with cancer progression and metastasis, including breast cancer. To date, most MDM2 inhibitors have been designed to block the MDM2-p53-binding interphase, and have low or no efficacy against advanced breast cancer with mutant or deficient p53. Here we use a high-throughput screening and computer-aided, structure-based rational drug design, and identify a lead compound, SP-141, which can directly bind to MDM2, inhibit MDM2 expression and induce its autoubiquitination and proteasomal degradation. SP-141 has strong in vitro and in vivo antibreast cancer activity, with no apparent host toxicity. While further investigation is needed, our data indicate that SP-141 is a novel targeted therapeutic agent that may especially benefit patients with advanced disease.

SummaryBreast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human data set analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases.

BACKGROUND The Eph family of receptor tyrosine kinases and their ephrin ligands are membrane-bound cell-signaling proteins and they play critical regulatory roles in embryonic development and carcinogenesis. Eph receptors require direct cell to cell interaction for activation and they are divided into EphA and EphB receptor classes, depending on their preferential binding affinity for EphrinA or EphrinB ligands. Eph receptors have been documented in breast cancer, but the Ephrin ligands have not been thoroughly investigated. MATERIALS AND METHODS We conducted a systematic assessment of EphrinB1 expression in a set of 75 formalin-fixed paraffin-embedded breast cancers, using immunohistochemical staining (IHC) with a specific antibody, and we examined the relationship between EphrinB1 expression, histopathological parameters, and the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. RESULTS High level expression of EphrinB1 is positively associated with lymph node metastasis (P < 0.0001) and with the presence of HER-2 receptor (P=0.041) and it is more often detected in triple-negative breast carcinomas (P=0.038). No relation was apparent between EphrinB1 expression level and other histopathological parameters, but enhanced EphrinB1 expression is associated with shorter overall survival (P=0.015). CONCLUSION Our analysis demonstrates that EphrinB1 expression is related to the metastasis of breast cancer and its enhanced expression confers a poor prognosis, suggesting that EphrinB1 may be a relevant therapeutic target in breast cancers.

Tamoxifen and aromatase inhibitors (AIs) have shown efficacy to prevent estrogen receptor positive (ER+) breast cancer; however, there is no effective prevention strategy for ER- breast cancer. HER2 overexpression (HER2+) can be detected in up to 40% of ER- breast cancers and it has been reported to promote the progression of early stage breast cancer. Here, we tested the feasibility of lapatinib to prevent ER-/HER2+ early stage breast disease. Our data showed that lapatinib reduced the proliferation and increased the apoptosis in an in vitro ER-/HER2+ early breast disease model. Furthermore, lapatinib treatment in the HER2-driven MMTV-neu mouse model from early stage of disease development (atypia, at 10 weeks of age) significantly delayed tumor initiation and progression as detected by mammary gland biopsy and fiber optic micro-endoscope (FOME) imaging. Mechanistically, we found that lapatinib inhibited GLUT4 upregulation and increased glucose up-taking in HER2-overexpressing cells. These results suggested that lapatinib is a promising agent for the prevention/intervention of ER- and HER2+ breast cancers. Our study brings better understanding of the mechanism underlying ER-/HER2+ early stage breast disease progression to cancer and our strategy of cancer prevention with low-dose kinase inhibitor could be rapidly translated into the clinic for other cancer types in the near future. Citation Format: Sunil Acharya, Jia Xu, Xiao Wang, Shalini Jain, Hai Wang, Qingling Zhang, Joseph Bower, Rebecca Richards-Kortum, Dihua Yu. Preventing the progression of estrogen receptor-negative/HER2-overexpressing early stage breast disease by lapatinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 832.

Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby facilitating cellular adaptation to the adverse environment and sustaining rapid proliferation. Recently, new evidence has emerged that abnormal metabolic reprogramming may occur at the early-stages of neoplasia and critically contribute to early transformation and cancer initiation. Previously, we reported that 14-3-3ζ overexpression is an early event in human breast cancer, taking place as early as in the atypical ductal hyperplasia (ADH) stage. We therefore hypothesized that 14-3-3ζ overexpression in breast epithelial cells may trigger metabolic reprogramming by elevating glycolysis that contributes to cancer initiation. In the present study, our bioinfomatics analysis of microarray profiling data from the early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of genes of the canonical glycolytic pathway, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human non-transformed mammary epithelial cells (hMECs), MCF10A and MCF12A, transcriptionally up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation of hMECs. Conversely, knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis by activating the MEK-ERK-CREB signaling axis and transcriptionally up-regulating LDHA, thereby critically contributes to human breast cancer initiation. Citation Format: Chia-Chi Chang, Chenyu Zhang, Ozgur Sahin, Qingling Zhang, Hai Wang, Jian Zhang, Sumaiyah Rehman, Ping Li, Dihua Yu. Upregulation of lactate dehydrogenase A by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-313.

Although ginseng and related herbs have a long history of utility for various health benefits, their application in cancer therapy and underlying mechanisms of action are not fully understood. Our recent work has shown that 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH(3)-PPD), a newly identified ginsenoside from Panax notoginseng, exerts activities against a variety of cancer cells in vitro and in vivo. This study was designed to investigate its anti-breast cancer activity and the underlying mechanisms of action. We observed that 25-OCH(3)-PPD decreased the survival of breast cancer cells by induction of apoptosis and G1 phase arrest and inhibited the growth of breast cancer xenografts in vivo. We further demonstrated that, in a dose- and time-dependent manner, 25-OCH(3)-PPD inhibited MDM2 expression at both transcriptional and post-translational levels in human breast cancer cells with various p53 statuses (wild type and mutant). Moreover, 25-OCH(3)-PPD inhibited in vitro cell migration, reduced the expression of epithelial-to-mesenchymal transition (EMT) markers, and prevented in vivo metastasis of breast cancer. In summary, 25-OCH(3)-PPD is a potential therapeutic and anti-metastatic agent for human breast cancer through down-regulating MDM2. Further preclinical and clinical development of this agent is warranted.

JKA97, a benzylidene analog of harmine, has been found to be a promising drug candidate for human cancer therapy, although the underlying molecular mechanisms have not been fully demonstrated. In this study, we evaluated the effects of JKA97 on human breast cancer cells in vitro and in vivo. JKA97 inhibited the growth and proliferation of MCF7 (p53 wild-type), MCF7 (p53 knockdown), and MDA-MB-468 (p53 mutant) cells in a dose-dependent manner. Treatment with JKA97 arrested breast cancer cells in G1 phase and induced apoptosis. JKA97 also significantly suppressed the growth of MCF7 and MDA-MB-468 xenograft tumors. It regulated the expression levels of G1 phase regulators, such as p21, p27, cyclinE, and cylinD1. JKA97 activated p21 transcription, independent of p53, but had little effect on p21 protein stability/degradation. In summary, our results suggest that JKA97 inhibits human breast cancer cell growth through activating p21, independent of p53, which provides a basis for developing this compound as a novel drug for human breast cancer therapy.

Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of CO2. CAIX is overexpressed in many types of cancer, including breast cancer, but is most frequently absent in corresponding normal tissues. CAIX expression is strongly induced by hypoxia and is significantly associated with tumor grade and poor survival. Herein, we show that hypoxia induces a significant increase in CAIX protein in MDA-MB-231 breast cancer cells. Using a unique mass spectrophotometric assay, we demonstrate that CAIX activity in plasma membranes isolated from MDA-MB-231 is correlated with CAIX content. We also show that CAIX exists predominantly as a dimeric, high-mannose N-linked glycoprotein. While there is some evidence that the dimeric form resides specifically in lipid rafts, our data do not support this hypothesis. EGF, alone, did not affect the distribution of CAIX into lipid rafts. However, acute EGF treatment in the context of hypoxia increased the amount of CAIX in lipid rafts by about 5-fold. EGF did not stimulate tyrosine phosphorylation of CAIX, although EGFR and down-stream signaling pathways were activated by EGF. Interestingly, hypoxia activated Akt independent of EGF action. Together, these data demonstrate that the active form of CAIX in the MDA-MB-231 breast cancer cell line is dimeric but that neither lipid raft localization nor phosphorylation are likely required for its dimerization or activity.

Contains fulltext : 80761.pdf (Publisher’s version ) (Closed access) The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a "triple-negative," basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydrase XII (CAXII). CAIX expression in the basal B cells is both density- and hypoxia-dependent and is correlated with carbonic anhydrase activity. Evidence is provided that CAIX contributes to extracellular acidification through studies on pH, lactic acid production, and CAIX inhibition. Together, these studies suggest that CAIX expression and activity is associated with metabolic dysfunction in MDA-MB-231 cells.

Twenty one human breast cancer lines were screened for expression of various RPTK including ErbB2, EGF, IGF-1 and insulin receptors. Those lines displayed a wide spectrum of colony forming and invasion activity. ErbB2 over expressing cells displayed an increased sensitivity toward inhibitors of the P13 kinase signaling pathway including rapamycin for monolayer and anchorage independent growth. Forced expression of ErbB2 in the non-ErbB2 over-expressing cells was able to confer the rapamycin hypersensitivity of those cells for anchorage independent growth. Rapamycin and herceptin additively inhibited adherent and anchorage independent growth of ErbB2 over-expressing breast cancer cells. An ErbB2 transgenic mouse model and the nude mouse xenograft model were used to test the tumor inhibitory effect of rapamycin and herceptin. Our result showed that rapamycin and herceptin each displayed a significant inhibition, and together demonstrated an additive inhibition, of tumor growth and tumor incidence, and thus combination of the two drugs could offer an improved anti-breast cancer therapy.

Breast cancer is one of the most commonly diagnosed cancers and the leading causes of cancer death in women worldwide. Natural product-oriented synthetic derivatives have played an important role in cancer drug discovery. JKA97, a benzylidene analog of harmine, is a novel anti-cancer candidate. The initial studies have shown its inhibitory effects on tumor growth in experimental models. However, the underlying molecular mechanisms are not fully elucidated. In this study, we determined the anti-cancer effects of JKA97 on human breast cancer in vitro and in vivo. Our data showed that, in a dose-dependent manner, JKA97 efficiently inhibited the growth and proliferation of human breast cancer cells with different p53 status, including MCF7 (p53 wild-type), MCF7 (p53 knockdown) and MDA-MB-468 (p53 mutant) cells. We also found that JKA97 induced apoptosis in human breast cancer cells independent of p53 and that JKA97 treatment resulted in G1 phase arrest in all the three breast cancer cell lines. In vivo, JKA97 significantly suppressed the growth of both breast cancer MCF7 and MDA-MB-468 xenograft tumors. Mechanistically, JKA97 modulated the levels of various cell-cycle regulators involved in G1 to S phase progression, such as p21, p27, cyclinE, and cyclinD1. Our results indicated that p21 and p27were up-regulated while cyclinE and cyclinD1 were down-regulated by JKA97 treatment. Using quantitative PCR and luciferase reporter assays, we demonstrated that the major mechanism for G1 phase arrest by JKA97 was induction of p21 at the transcriptional level, independent of p53. We further confirmed the role of p21 in JKA97-induced G1 phase arrest by using HCT116 cells with or without p21 expression. Taken together, these findings suggest that JKA97 inhibits human breast cancer by inducing cell cycle arrest through up-regulation of p21, regardless of p53 status. Considering that more than 50% human cancers have no or mutant p53 expression, our results provide a basis for further development of this compound as potential drug for breast cancer therapy. (This work was supported in part by NIH/NCI grants R01 CA112029 and R01 CA121211, R15 CA100102 and a grant (BCTR070731) from Susan G Komen for the Cure.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3834. doi:1538-7445.AM2012-3834

ISG15 (interferon-stimulated gene 15), an ubiquitin-like protein (UBL), is a new tumor marker whose expression has been correlated with disease progression in bladder cancer and suggested to be prognostic for breast cancer. However, the molecular basis for, and the function of, ISG15 overexpression in tumors remain unclear. Here, we show that ISG15 overexpression in both breast cancer cells and oncogenic Ras-transformed mammary epithelial cells (MCF-10A) is due to elevated secretion of interferon-β. Moreover, interferon-β-ISG15 autocrine signaling in oncogenic Ras-transformed MCF-10A cells results in increased colony formation efficiency. Interestingly, gain of epithelial-mesenchymal transition (EMT) characteristics, including appearance of spindle-shaped cell morphology and EMT markers, as well as increased cell migration, in Ras-transformed MCF-10A cells is also dependent on interferon-β-ISG15 autocrine signaling. Our results demonstrate for the first time that tumor cell-secreted interferon-β may modulate tumor cell growth and migration through ISG15, suggesting potential cancer therapeutic approaches by antagonizing interferon autocrine signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3438. doi:10.1158/1538-7445.AM2011-3438