Oncogenic signaling in amphiregulin and EGFR‐expressing PTEN‐null human breast cancer

A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these determinants have a poor prognosis. We used cell line models of EGFR‐positive/PTEN null TNBC to elucidate the signaling networks that drive the malignant features of these cells and cause resistance to EGFR inhibitors. In these cells, amphiregulin (AREG)‐mediated activation of EGFR results in up‐regulation of fibronectin (FN1), which is known to be a mediator of invasive capacity via interaction with integrin β1. EGFR activity in this PTEN null background also results in Wnt/beta‐catenin signaling and activation of NF‐κB. In addition, AKT is constitutively phosphorylated in these cells and is resistant to gefitinib. Expression profiling demonstrated that AREG‐activated EGFR regulates gene expression differently than EGF‐activated EGFR, and functional analysis via genome‐scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are essential for survival of SUM‐149 cells, but are uncoupled from EGFR signaling. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, which likely mediates resistance to EGFR inhibitors.     

TRAIL induces apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in some but not all breast cancer cell lines. Breast cancers can be divided into those which express the estrogen (ER) and progesterone (PR) receptors, those with HER-2 amplification, and those without expression of ER, PR, or HER-2 amplification (referred to as basal or triple-negative breast cancer). We tested a panel of 20 breast cancer cell lines representing the different types of breast cancer to evaluate if the molecular phenotype of the breast cancer cells determined their response to TRAIL. The most striking finding was that eight of eleven triple-negative cell lines are sensitive to TRAIL-mediated apoptosis. The eight TRAIL-sensitive triple-negative cell lines have a mesenchymal phenotype while the three TRAIL-resistant triple-negative cell lines have an epithelial phenotype. Two of five cell lines with HER-2 amplification were sensitive to TRAIL and none of the five ER positive cell lines were sensitive. RNAi-mediated knockdown of TRAIL receptor expression demonstrated that TRAIL Receptor 2 (TRAIL-R2) mediates the effects of TRAIL, even when both TRAIL-R1 and TRAIL-R2 are expressed. Finally, inhibition of EGFR, expressed in both TRAIL-sensitive and TRAIL-resistant triple-negative breast cancer cell lines, using a small molecule tyrosine kinase inhibitor (AG1478), enhanced TRAIL-induced apoptosis in TRAIL-sensitive cell lines but did not convert resistant cells into TRAIL-sensitive cells. Together, these findings suggest that a subset of triple-negative breast cancer, those with mesenchymal features, may be the most likely to benefit from TRAIL targeted therapy. These findings could form the basis to select breast cancer patients for clinical trials of TRAIL-R2 ligands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. R. Davis and J. G. Pumphrey contributed equally to this work.     

Intercellular adhesion molecule‐1‐targeted near‐infrared photoimmunotherapy of triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and conventional chemotherapy and molecular‐targeted therapies show limited efficacy. Near‐infrared photoimmunotherapy (NIR‐PIT) is a new anticancer treatment that selectively damages the cell membrane of cancer cells based on NIR light‐induced photochemical reactions of the antibody (Ab)‐photoabsorber (IRDye700Dx) conjugate and the cell membrane. TNBC is known to express several adhesion molecules on the cell surface providing a potential new target for therapy. Here, we investigated the therapeutic efficacy of intercellular adhesion molecule‐1 (ICAM‐1)‐targeted NIR‐PIT using xenograft mouse models subcutaneously inoculated with two human ICAM‐1‐expressing TNBC cell lines, MDAMB468‐luc and MDAMB231 cells. In vitro ICAM‐1‐targeted NIR‐PIT damaged both cell types in a NIR light dose‐dependent manner. In vivo ICAM‐1‐targeted NIR‐PIT in both models showed early histological signs of cancer cell damage, such as cytoplasmic vacuolation. Even among the cancer cells that appeared to be morphologically intact within 2 h post treatment, abnormal distribution of the actin cytoskeleton and a significant decrease in Ki‐67 positivity were observed, indicating widespread cellular injury reflected in cytoplasmic degeneration. Such damage to cancer cells by NIR‐PIT significantly inhibited subsequent tumor growth and improved survival. This study suggests that ICAM‐1‐targeted NIR‐PIT could have potential clinical application in the treatment of TNBC.     

Autophagy activation and SREBP‐1 induction contribute to fatty acid metabolic reprogramming by leptin in breast cancer cells

Leptin, a hormone predominantly derived from adipose tissue, is well known to induce growth of breast cancer cells. However, its underlying mechanisms remain unclear. In this study, we examined the role of reprogramming of lipid metabolism and autophagy in leptin‐induced growth of breast cancer cells. Herein, leptin induced significant increase in fatty acid oxidation‐dependent ATP production in estrogen receptor‐positive breast cancer cells. Furthermore, leptin induced both free fatty acid release and intracellular lipid accumulation, indicating a multifaceted effect of leptin in fatty acid metabolism. These findings were further validated in an MCF‐7 tumor xenograft mouse model. Importantly, all the aforementioned metabolic effects of leptin were mediated via autophagy activation. In addition, SREBP‐1 induction driven by autophagy and fatty acid synthase induction, which is mediated by SREBP‐1, plays crucial roles in leptin‐stimulated metabolic reprogramming and are required for growth of breast cancer cell, suggesting a pivotal contribution of fatty acid metabolic reprogramming to tumor growth by leptin. Taken together, these results highlighted a crucial role of autophagy in leptin‐induced cancer cell‐specific metabolism, which is mediated, at least in part, via SREBP‐1 induction.

Abbreviations

2‐DG

2‐deoxyglucose

3‐MA

3‐methyladenine

ACC‐1

acetyl‐CoA carboxylase 1

ACLY

ATP citrate lyase

ER

estrogen receptor

FADS1

fatty acid desaturase 1

FADS2

fatty acid desaturase 2

FAO

fatty acid oxidation

FAS

fatty acid synthesis

FASN

fatty acid synthase

FFA

free fatty acid

IHC

immunohistochemistry

SCD‐1

stearoyl‐CoA desaturase‐1

SREBP‐1

sterol regulatory element‐binding protein 1

     

Upregulation of S100A10 in metastasized breast cancer stem cells

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44⁺ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44⁺ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell–related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.     

Autophagy inhibits cancer stemness in triple‐negative breast cancer via miR‐181a‐mediated regulation of ATG5 and/or ATG2B

Autophagy has a dual role in the maintenance of cancer stem cells (CSCs), but the precise relationship between autophagy and cancer stemness requires further investigation. In this study, it was found that luminal and triple‐negative breast cancers require distinct therapeutic approaches because of their different amounts of autophagy flux. We identified that autophagy flux was inhibited in triple‐negative breast cancer (TNBC) CSCs. Moreover, miRNA‐181a (miR‐181a) expression is upregulated in both TNBC CSCs and patient tissues. Autophagy‐related 5 (ATG5) and autophagy‐related 2B (ATG2B) participate in the early formation of autophagosomes and were revealed as targets of miR‐181a. Inhibition of miR‐181a expression led to attenuation of TNBC stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. In summary, autophagy inhibits cancer stemness through the miR‐181a‐regulated mechanism in TNBC. Promoting tumor‐suppressive autophagy using curcumin may be a potential method for the treatment of TNBC.     

Cytotoxicity, apoptosis induction and anti-metastatic potential of Oroxylum indicum in human breast cancer cells

Despite clinical advances in anticancer therapy, there is still a need for novel anticancer metabolites, with higher efficacy and lesser side effects. Oroxylum indicum (L.) Vent. is a small tree of the Bignoniaceae family which is well known for its food and medicinal properties. In present study, the chemopreventive properties of O. indicum hot and cold non-polar extracts (petroleum ether and chloroform) were investigated with MDA- MB-231 (cancer cells) and WRL-68 (non-tumor cells) by XTT assay. All the extracts, and particularly the petroleum ether hot extract (PHO), exhibited significantly (P<0.05) higher cytotoxicity in MDA-MB-231 when compared to WRL-68 cells. PHO was then tested for apoptosis induction in estrogen receptor (ER)-negative (MDA-MB-231) and ER-positive (MCF-7) breast cancer cells by cellular DNA fragmentation ELISA, where it proved more efficient in the MDA-MB-231 cells. Further, when PHO was tested for anti-metastatic potential in a cell migration inhibition assay, it exhibited beneficial effects. Thus non-polar extracts of O. indicum (especially PHO) can effectively target ER-negative breast cancer cells to induce apoptosis, without harming normal cells by cancer-specific cytotoxicity. Hence, it could be considered as an extract with candidate precursors to possibly harness or alleviate ER-negative breast cancer progression even in advanced stages of malignancy.     

西达本胺对三阴性乳腺癌细胞凋亡和侵袭的影响

目的 探讨组蛋白去乙酰化酶抑制剂（HDACi）西达本胺对三阴性乳腺癌细胞株CAL 51体外增殖、凋亡及侵袭的作用。方法 采用实时无标记细胞分析（RTCA）技术检测不同浓度西达本胺（0、5、10、20、50、100μmol／L）对CAL 51细胞的增殖抑制作用;倒置显微镜下观察西达本胺（10、15、20μmol／L）处理CAL 51细胞72h后的细胞形态;流式细胞仪检测细胞周期、凋亡及乳腺癌干细胞比例;采用RTCA技术检测西达本胺对细胞侵袭、浸润能力的影响。结果 西达本胺在体外能明显抑制CAL 51细胞的增殖,且与药物浓度呈正相关（r=0.791,P＜0.001）;细胞形态发生改变;流式细胞仪检测显示细胞出现明显凋亡,且随药物浓度、作用时间的增加而升高（P＜0.05）,细胞周期和干细胞比例无变化;西达本胺对CAL-51细胞的侵袭、浸润能力有抑制作用,且随着药物浓度的增加,细胞侵袭能力明显降低（P＜0.05）。结论 西达本胺能够明显抑制CAL-51细胞的生长,诱导其凋亡,并降低其侵袭、浸润能力。     

Predicting dynamic response to neoadjuvant chemotherapy in breast cancer: a novel metabolomics approach

Neoadjuvant chemotherapy (NACT) outcomes vary according to breast cancer (BC) subtype. Since pathologic complete response is one of the most important target endpoints of NACT, further investigation of NACT outcomes in BC is crucial. Thus, identifying sensitive and specific predictors of treatment response for each phenotype would enable early detection of chemoresistance and residual disease, decreasing exposures to ineffective therapies and enhancing overall survival rates. We used liquid chromatography−high‐resolution mass spectrometry (LC‐HRMS)‐based untargeted metabolomics to detect molecular changes in plasma of three different BC subtypes following the same NACT regimen, with the aim of searching for potential predictors of response. The metabolomics data set was analyzed by combining univariate and multivariate statistical strategies. By using ANOVA–simultaneous component analysis (ASCA), we were able to determine the prognostic value of potential biomarker candidates of response to NACT in the triple‐negative (TN) subtype. Higher concentrations of docosahexaenoic acid and secondary bile acids were found at basal and presurgery samples, respectively, in the responders group. In addition, the glycohyocholic and glycodeoxycholic acids were able to classify TN patients according to response to treatment and overall survival with an area under the curve model > 0.77. In relation to luminal B (LB) and HER2+ subjects, it should be noted that significant differences were related to time and individual factors. Specifically, tryptophan was identified to be decreased over time in HER2+ patients, whereas LysoPE (22:6) appeared to be increased, but could not be associated with response to NACT. Therefore, the combination of untargeted‐based metabolomics along with longitudinal statistical approaches may represent a very useful tool for the improvement of treatment and in administering a more personalized BC follow‐up in the clinical practice.     

Loss of GABARAPL1 confers ferroptosis resistance to cancer stem‐like cells in hepatocellular carcinoma

Cancer stem‐like cells (CSLC) are considered a major contributor to the development and progression of hepatocellular carcinoma (HCC). Previous studies indicated that CSLC are characterized by resistance to ferroptosis, a type of lipid peroxidation‐dependent cell death. Here, we identified a set of ferroptosis‐related stemness genes (FRSG) and found that these genes may be involved in immune infiltration in HCC. A four‐FRSG (CDKN2A, GABARAPL1, HRAS, RPL8) risk model with prognostic prediction was constructed by a Cox analysis in HCC. Among these four genes, GABARAPL1 was downregulated in HCC tumor‐repopulating cells (TRC; a type of CSLC). Its downregulation decreased the sensitivity of HCC TRC to erastin‐ or sorafenib‐triggered ferroptosis. Together, we uncovered a molecular mechanism via which CSLC could achieve tolerance to ferroptosis. Further studies may provide potential therapeutic strategies targeting CSLC in HCC.     

The ELEANOR noncoding RNA expression contributes to cancer dormancy and predicts late recurrence of estrogen receptor‐positive breast cancer

The recurrence risk of estrogen receptor (ER)‐positive breast cancer remains high for a long period of time, unlike other types of cancer. Late recurrence reflects the ability of cancer cells to remain dormant through various events, including cancer stemness acquisition, but the detailed mechanism is unknown. ESR1 locus enhancing and activating noncoding RNAs (ELEANORS) are a cluster of nuclear noncoding RNAs originally identified in a recurrent breast cancer cell model. Although their functions as chromatin regulators in vitro are well characterized, their roles in vivo remain elusive. In this study, we evaluated the clinicopathologic features of ELEANORS, using primary and corresponding metastatic breast cancer tissues. The ELEANOR expression was restricted to ER‐positive cases and well‐correlated with the ER and progesterone receptor expression levels, especially at the metastatic sites. ELEANORS were detected in both primary and metastatic tumors (32% and 29%, respectively), and frequently in postmenopausal cases. Interestingly, after surgery, patients with ELEANOR‐positive primary tumors showed increased relapse rates after, but not within, 5 years. Multivariate analysis showed that ELEANORS are an independent recurrence risk factor. Consistently, analyses with cell lines, mouse xenografts, and patient tissues revealed that ELEANORS upregulate a breast cancer stemness gene, CD44, and maintain the cancer stem cell population, which could facilitate tumor dormancy. Our findings highlight a new role of nuclear long noncoding RNAs and their clinical potential as predictive biomarkers and therapeutic targets for late recurrence of ER‐positive breast cancer.     

Gene expression profiles of breast cancer metastasis according to organ site

In advanced breast cancer, biomarker identification and patient selection using a metastatic tumor biopsy is becoming more necessary. However, the biology of metastasis according to the organ site is largely unknown. Here, we evaluated the expression of 771 genes in 184 metastatic samples across 11 organs, including liver, lung, brain, and bone, and made the following observations. First, all PAM50 molecular intrinsic subtypes were represented across organs and within immunohistochemistry‐based groups. Second, HER2‐low disease was identified across all organ sites, including bone, and HER2 expression significantly correlated with ERBB2 expression. Third, the majority of expression variation was explained by intrinsic subtype and not organ of metastasis. Fourth, subtypes and individual subtype‐related genes/signatures were significantly associated with overall survival. Fifth, we identified 74 genes whose expression was organ‐specific and subtype‐independent. Finally, immune profiles were found more expressed in lung compared to brain or liver metastasis. Our results suggest that relevant tumor biology can be captured in metastatic tissues across a variety of organ sites; however, unique biological features according to organ site were also identified and future studies should explore their implications in diagnostic and therapeutic interventions.     

Association between germline pathogenic variants and breast cancer risk in Japanese women: The HERPACC study

Approximately 5%–10% of breast cancers are hereditary, caused by germline pathogenic variants (GPVs) in breast cancer predisposition genes. To date, most studies of the prevalence of GPVs and risk of breast cancer for each gene based on cases and noncancer controls have been conducted in Europe and the United States, and little information from Japanese populations is available. Furthermore, no studies considered confounding by established environmental factors and single‐nucleotide polymorphisms (SNPs) identified in genome‐wide association studies (GWAS) together in GPV evaluation. To evaluate the association between GPVs in nine established breast cancer predisposition genes including BRCA1/2 and breast cancer risk in Japanese women comprehensively, we conducted a case‐control study within the Hospital‐based Epidemiologic Research Program at Aichi Cancer Center (629 cases and 1153 controls). The associations between GPVs and the risk of breast cancer were assessed by odds ratios (OR) and 95% confidence intervals (CI) using logistic regression models adjusted for potential confounders. A total of 25 GPVs were detected among all cases (4.0%: 95% CI: 2.6–5.9), whereas four individuals carried GPVs in all controls (0.4%). The OR for breast cancer by all GPVs and by GPVs in BRCA1/2 was 12.2 (4.4–34.0, p = 1.74E‐06) and 16.0 (4.2–60.9, p = 5.03E‐0.5), respectively. A potential confounding with GPVs was observed for the GWAS‐identified SNPs, whereas not for established environmental risk factors. In conclusion, GPVs increase the risk of breast cancer in Japanese women regardless of environmental factors and GWAS‐identified SNPs. Future studies investigating interactions with environment and SNPs are warranted.     

Ultradeep targeted sequencing of circulating tumor DNA in plasma of early and advanced breast cancer

We present a study to evaluate the feasibility and clinical utility of amplicon‐based Oncomine Pan‐Cancer cell‐free assay to detect circulating tumor DNA (ctDNA) in patients with early or advanced breast cancer. In this study, 109 early and metastatic breast cancer patients were recruited before the initiation of treatment. ctDNA mutation profiles were assessed through unique molecular tagging (UMT) and ultradeep next generation sequencing (NGS). For patients with mutations, DNA from corresponding white blood cells (WBC) was sequenced to exclude variants of clonal‐hematopoietic (CH) origin. UMT targeted sequencing from plasma of 109 patients achieved a median total coverage of 55 498X and a median molecular coverage of 4187X. Among 53 ctDNA positive samples, 38% were mutation positive by WBC sequencing, indicating potentially false‐positive results contributed by CH origin. Prevalence of CH‐related mutations was associated with age (P = 7.51 × 10⁻⁴). After exclusion of CH mutations, ctDNA detection rates were 37% for local or locally advanced breast cancer (stage I‐III) and 81% for metastatic or recurrent breast cancer. The ctDNA detection rate correlated with disease stage (P = 2.60 × 10⁻⁴), nodal spread (P = 6.49 × 10⁻³) and the status of distant metastases (P = 5.00 × 10⁻⁴). ctDNA variants were detected mostly in TP53, PIK3CA and AKT1 genes, with variants showing therapeutic relevance. This pilot study endorses the use of targeted NGS for non‐invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.