Store-independent activation of Orai1 by SPCA2 in mammary tumors.

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.

Intraductal administration of transferrin receptor-targeted immunotoxin clears ductal carcinoma in situ in mouse models of breast cancer-a preclinical study.

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 µg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 µg/teat). MCF7 tumor-bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

Multiple roles of HOX proteins in Metastasis: Let me count the ways.

Knowledge of the role of HOX proteins in cancer has been steadily accumulating in the last 25 years. They are encoded by 39 HOX genes arranged in 4 distinct clusters, and have unique and redundant function in all types of cancers. Many HOX genes behave as oncogenic transcriptional factors regulating multiple pathways that are critical to malignant progression in a variety of tumors. Some HOX proteins have dual roles that are tumor-site specific, displaying both oncogenic and tumor suppressor function. The focus of this review is on how HOX proteins contribute to growth or suppression of metastasis. The review will cover HOX protein function in the critical aspects of epithelial-mesenchymal transition, in cancer stem cell sustenance and in therapy resistance, manifested as distant metastasis. The emerging role of adiposity in both initiation and progression of metastasis is described. Defining the role of HOX genes in the metastatic process has identified candidates for targeted cancer therapies that may combat the metastatic process. We will discuss potential therapeutic opportunities, particularly in pathways influenced by HOX proteins.

Intraductal Therapy in Breast Cancer: Current Status and Future Prospective.

With our improved understanding of the biological behavior of breast cancer, minimally invasive intervention is urgently needed for personalized treatment of early disease. Intraductal therapy is one such minimally invasive approach. With the help of appropriate tools, technologies using the intraductal means of entering the ducts may be used both to diagnose and treat lesions in the mammary duct system with less trauma and at the same time avoid systemic toxicity. Traditional agents such as those targeting pathways, endocrine therapy, immunotherapy, or gene therapy can be used alone or combined with other new technologies, such as nanomaterials, through the intraductal route. Additionally, relevant mammary tumor models in rodents which reflect changes in the tumor microenvironment will help deepen our understanding of their biological behavior and heterogeneity. This article reviews the current status and future prospects of intraductal therapy in breast cancer, with emphasis on ductal carcinoma in situ.

Unpredicted central inversion in a sgRNA flanked by inverted repeats.

In genome engineering, sgRNAs define the genomic target to be modified in CRISPR/Cas9 system. Either for single gene editing or genome-wide screens, sgRNAs are cloned into plasmid vectors. During our performance of CRISPR/Cas9 gene knock out, we found that the central part of a sgRNA was inverted after transformation into Escherichia coli. Interestingly, the inverted portion was found to be flanked by inverted repeats, and sealing of nicks inside the plasmid could correct the inversion. This type of sgRNA recombination completely changed its original sequence and should be noted during sgRNA design and performance of CRISPR/Cas9.

Intraductal fulvestrant for therapy of ERα-positive ductal carcinoma in situ of the breast: a preclinical study.

Mammographic screening for breast cancer has led to increased detection of ductal carcinoma in situ (DCIS) and a reappraisal of the necessity of aggressive treatment with their attendant toxicities for a preneoplastic lesion. Fulvestrant, a selective estrogen receptor degrader, is very effective in the treatment of estrogen receptor positive (ER+) breast cancer, but delivery by the painful intramuscular (i.m) route is limiting. We hypothesized that intraductal (i.duc) administration of fulvestrant will provide a direct, safe and effective treatment for DCIS. Mice bearing mammary ductal xenografts of ER+, luciferase-tagged MCF-7 breast cancer cells were administered vehicle or fulvestrant i.m or i.duc. I.duc MCF-7-luc tumors in mice treated with fulvestrant i.duc or i.m grew significantly slower than vehicle control. Whole mount analysis and histopathology showed that i.duc fulvestrant achieved significantly larger cancer-free areas. Western blot analysis showed reduced levels of estrogen receptor alpha (ERα) and its downstream targets, c-Myc and Cyclin D1, and increased levels of ERß, which is known to inhibit ERα function. Immunohistochemical analysis of tumor sections showed that Ki67 and ERα protein levels decreased by 3-fold, and neoangiogenesis was inhibited by i.duc fulvestrant treatment. I.duc fulvestrant also reduced outgrowth of ERα+, autochthonous N-methyl-N-nitrosourea-induced mammary tumors in rats. Overall, we have shown that i.duc fulvestrant was significantly more effective than, or equivalent in action to i.m fulvestrant in two preclinical models of breast cancer. These studies provide evidence for a novel and safe route for fulvestrant therapy of DCIS and prevention of breast cancer. This preclinical study provides a strong basis for conducting clinical trials for DCIS and early breast cancer.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays.

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Induction of cell cycle arrest and inflammatory genes by combined treatment with epigenetic, differentiating, and chemotherapeutic agents in triple-negative breast cancer.

BACKGROUND: A combination of entinostat, all-trans retinoic acid, and doxorubicin (EAD) induces cell death and differentiation and causes significant regression of xenografts of triple-negative breast cancer (TNBC). METHODS: We investigated the mechanisms underlying the antitumor effects of each component of the EAD combination therapy by high-throughput gene expression profiling of drug-treated cells. RESULTS: Microarray analysis showed that entinostat and doxorubicin (ED) altered expression of genes related to growth arrest, inflammation, and differentiation. ED downregulated MYC, E2F, and G2M cell cycle genes. Accordingly, entinostat sensitized the cells to doxorubicin-induced growth arrest at G2. ED induced interferon genes, which correlated with breast tumors containing a higher proportion of tumor-infiltrating lymphocytes. ED also increased the expression of immune checkpoint agonists and cancer testis antigens. Analysis of TNBC xenografts showed that EAD enhanced the inflammation score in nude mice. Among the genes differentially regulated between the EAD and ED groups, an all-trans retinoic acid (ATRA)-regulated gene, DHRS3, was induced in EAD-treated xenografts. DHRS3 was expressed at lower levels in human TNBC metastases compared to normal breast or primary tumors. High expression of ED-induced growth arrest and inflammatory genes was associated with better prognosis in TNBC patients. CONCLUSIONS: Entinostat potentiated doxorubicin-mediated cell death and the combination induced inflammatory signatures. The ED-induced immunomodulation may improve immunotherapy. Addition of ATRA to ED may potentiate inflammation and contribute to TNBC regression.

Tumor and serum DNA methylation in women receiving preoperative chemotherapy with or without vorinostat in TBCRC008.

BACKGROUND: Methylated gene markers have shown promise in predicting breast cancer outcomes and treatment response. We evaluated whether baseline and changes in tissue and serum methylation levels would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer undergoing preoperative chemotherapy. METHODS: The TBCRC008 trial investigated pCR following 12 weeks of preoperative carboplatin and albumin-bound paclitaxel + vorinostat/placebo (n = 62). We measured methylation of a 10-gene panel by quantitative multiplex methylation-specific polymerase chain reaction and expressed results as cumulative methylation index (CMI). We evaluated association between CMI level [baseline, day 15 (D15), and change] and pCR using univariate and multivariable logistic regression models controlling for treatment and hormone receptor (HR) status, and performed exploratory subgroup analyses. RESULTS: In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with 40% lower chance of obtaining pCR (odds ratio, OR 0.60, 95% CI 0.37-0.97; p = 0.037). Subgroup analyses suggested a significant association between tissue D15 CMI levels and pCR in vorinostat-treated [OR 0.44 (0.20, 0.93), p = 0.03], but not placebo-treated patients. CONCLUSION: In this study investigating the predictive roles of tissue and serum CMI levels in patients with early breast cancer for the first time, we demonstrate that high D15 tissue CMI levels may predict poor response. Larger studies and improved analytical procedures to detect methylated gene markers in early stage breast cancer are needed. TBCRC008 is registered on ClinicalTrials.gov (NCT00616967).

Correction to: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

[This corrects the article DOI: 10.1186/s12014-018-9197-x.].

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. METHODS: In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. RESULTS: We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. CONCLUSIONS: Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

HOX genes: Major actors in resistance to selective endocrine response modifiers.

Long term treatment with therapies aimed at blocking the estrogen- (ER) or androgen receptor (AR) action often leads to the development of resistance to selective modulators of the estrogen receptor (SERMs) in ERα-positive breast cancer, or of the androgen receptor (SARMs) in AR-positive prostate cancer. Many underlying molecular events that confer resistance are known, but a unifying theme is yet to be revealed. Receptor tyrosine kinases (RTKs) such EGFR, ERBB2 and IGF1R are major mediators that can directly alter cellular response to the SERM, tamoxifen, but the mechanisms underlying increased expression of RTKs are not clear. A number of HOX genes and microRNAs and non-coding RNAs residing in the HOX cluster, have been identified as important independent predictors of endocrine resistant breast cancer. Recently, convincing evidence has accumulated that several members belonging to the four different HOX clusters contribute to endocrine therapy resistant breast cancer, but the mechanisms remain obscure. In this article, we have reviewed recent progress in understanding of the functioning of HOX genes and regulation of their expression by hormones. We also discuss, in particular, the contributions of several members of the HOX gene family to endocrine resistant breast cancer.

Guidelines for the selection of functional assays to evaluate the hallmarks of cancer.

The hallmarks of cancer capture the most essential phenotypic characteristics of malignant transformation and progression. Although numerous factors involved in this multi-step process are still unknown to date, an ever-increasing number of mutated/altered candidate genes are being identified within large-scale cancer genomic projects. Therefore, investigators need to be aware of available and appropriate techniques capable of determining characteristic features of each hallmark. We review the methods tailored to experimental cancer researchers to evaluate cell proliferation, programmed cell death, replicative immortality, induction of angiogenesis, invasion and metastasis, genome instability, and reprogramming of energy metabolism. Selecting the ideal method is based on the investigator's goals, available equipment and also on financial constraints. Multiplexing strategies enable a more in-depth data collection from a single experiment - obtaining several results from a single procedure reduces variability and saves time and relative cost, leading to more robust conclusions compared to a single end point measurement. Each hallmark possesses characteristics that can be analyzed by immunoblot, RT-PCR, immunocytochemistry, immunoprecipitation, RNA microarray or RNA-seq. In general, flow cytometry, fluorescence microscopy, and multiwell readers are extremely versatile tools and, with proper sample preparation, allow the detection of a vast number of hallmark features. Finally, we also provide a list of hallmark-specific genes to be measured in transcriptome-level studies. Although our list is not exhaustive, we provide a snapshot of the most widely used methods, with an emphasis on methods enabling the simultaneous evaluation of multiple hallmark features.

Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.

Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.

Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

Molecular definition of breast tumor heterogeneity.

Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells purified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations.

Altered antisense-to-sense transcript ratios in breast cancer.

Transcriptome profiling studies suggest that a large fraction of the genome is transcribed and many transcripts function independent of their protein coding potential. The relevance of noncoding RNAs (ncRNAs) in normal physiological processes and in tumorigenesis is increasingly recognized. Here, we describe consistent and significant differences in the distribution of sense and antisense transcripts between normal and neoplastic breast tissues. Many of the differentially expressed antisense transcripts likely represent long ncRNAs. A subset of genes that mainly generate antisense transcripts in normal but not cancer cells is involved in essential metabolic processes. These findings suggest fundamental differences in global RNA regulation between normal and cancer cells that might play a role in tumorigenesis.

The HOXB7 protein renders breast cancer cells resistant to tamoxifen through activation of the EGFR pathway.

Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.

Combining the strength of genomics, nanoparticle technology, and direct intraductal delivery for breast cancer treatment.

A large number of genes are altered in cancer cells. Often, reversal or inhibition of just one of these alterations leads to death of the cancer cells. Technological advances in multiple areas are necessary to potentiate clinical translation of these findings. In a recent article, Brock and colleagues reported that overexpressed HOXA1 is a critical event in tumor progression in a mouse mammary tumor model. They developed HOXA1-small interfering RNA nanoparticles and achieved effective therapeutic doses by delivering them intraductally through the nipple to the site of the tumor and at the same time circumvented the systemic immune response. This study strengthens the concept of targeting overexpressed genes by using small interfering RNA and bypassing systemic immunity through local intraductal delivery.

Subtype-specific overexpression of the Rac-GEF P-REX1 in breast cancer is associated with promoter hypomethylation.

INTRODUCTION: The Rac-GEF P-REX1 is a key mediator of ErbB signaling in breast cancer recently implicated in mammary tumorigenesis and metastatic dissemination. Although P-REX1 is essentially undetectable in normal human mammary epithelial tissue, this Rac-GEF is markedly upregulated in human breast carcinomas, particularly of the luminal subtype. The mechanisms underlying P-REX1 upregulation in breast cancer are unknown. Toward the goal of dissecting the mechanistic basis of P-REX1 overexpression in breast cancer, in this study we focused on the analysis of methylation of the PREX1 gene promoter. METHODS: To determine the methylation status of the PREX1 promoter region, we used bisulfite genomic sequencing and pyrosequencing approaches. Re-expression studies in cell lines were carried out by treatment of breast cancer cells with the demethylating agent 5-aza-2'-deoxycitidine. PREX1 gene methylation in different human breast cancer subtypes was analyzed from the TCGA database. RESULTS: We found that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and that DNA methylation in this region inversely correlates with P-REX1 expression in human breast cancer cell lines. A comprehensive analysis of human breast cancer cell lines and tumors revealed significant hypomethylation of the PREX1 promoter in ER-positive, luminal subtype, whereas hypermethylation occurs in basal-like breast cancer. Treatment of normal MCF-10A or basal-like cancer cells, MDA-MB-231 with the demethylating agent 5-aza-2'-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A restores P-REX1 levels to those observed in luminal breast cancer cell lines, suggesting that aberrant expression of P-REX1 in luminal breast cancer is a consequence of PREX1 promoter demethylation. Unlike PREX1, the pro-metastatic Rho/Rac-GEF, VAV3, is not regulated by methylation. Notably, PREX1 gene promoter hypomethylation is a prognostic marker of poor patient survival. CONCLUSIONS: Our study identified for the first time gene promoter hypomethylation as a distinctive subtype-specific mechanism for controlling the expression of a key regulator of Rac-mediated motility and metastasis in breast cancer.