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Objective: To determine the added value of digital breast tomosynthesis (DBT) in the assessment of lesions detected by contrast-enhanced mammography (CEM)., Material and Methods: A retrospective study was conducted in a tertiary university medical center. All CEM studies including DBT performed between January 2016 and December 2020 were included. Lesions were categorized and scored by four dedicated breast radiologists according to the recent CEM and DBT supplements to the Breast Imaging Reporting and Data System (BIRADS) lexicon. Changes in the BIRADS score of CEM-detected lesions with the addition of DBT were evaluated according to the pathology results and 1-year follow-up imaging study., Results: BIRADS scores of CEM-detected lesions were upgraded toward the lesion's pathology with the addition of DBT ( p > 0.0001), overall and for each reader. The difference in BIRADS scores before and after the addition of DBT was more significant for readers who were less experienced. The reason for changes in the BIRADS score was better lesion margin visibility. The main BIRADS descriptors applied in the malignant lesions were spiculations, calcifications, architectural distortion, and sharp or obscured margins., Conclusions: The addition of DBT to CEM provides valuable information on the enhancing lesion, leading to a more accurate BIRADS score.

Objectives: High background parenchymal enhancement (BPE) levels and asymmetric distribution could cause diagnostic uncertainty due to morphological similarity to breast cancer, especially invasive lobular carcinoma (ILC). We investigated BPE in ILC patients, its association with the tumor hormonal profile, and the effect of endocrine treatment (ET)., Methods: The analysis included all MRI examinations performed at our institution between 2010 and 2019 for ILC-diagnosed patients. Baseline study and the first follow-up study were reviewed. Digital medical records were reviewed to retrieve demographics/pathology results/treatment information. BPE and fibroglandular tissue were assessed qualitatively on the contralateral breast according to the criteria of the Breast Imaging Reporting and Data System (BI-RADS)., Results: The study included 129 patients. Most (91%) had pure ILC. All received ET; 12% also received chemotherapy; 90% had surgery first; 70% by breast conservation. On the baseline MRI, 70% had mild or moderate BPE; whereas, on the follow-up study, the majority (59%) had minimal BPE. Most BPE reductions were by 2 degrees. In the baseline study, additional biopsies were required in 59% of cases, and in 17%, a short-term follow-up was recommended. In the follow-up study, biopsies were recommended in 10%, and a short-term follow-up was requested in 16%. A correlation between progesterone receptor intensity index and baseline BPE level was observed (r = 0.3, p = 0.004)., Conclusion: ILC patients usually exhibit high BPE. ET decreases BPE, and therefore may decrease false-positive interpretations. Additional research is needed to explore whether study can be performed on ET without compromising sensitivity., Key Points: ∙ High background parenchymal enhancement levels reduces breast MRI sensitivity, yielding high false positive rates especially when reporting cases of invasive lobular carcinoma [ILC].∙Treatment of ILC with endocrine therapy reduces background parenchymal enhancement and thus could decrease these false-positive interpretations.

Neoadjuvant therapy (NAT) is increasingly used in breast cancer (BC), yet, the recommended time interval between NAT completion, preoperative imaging assessment, and breast surgery is not clearly defined. This single-center retrospective study investigated tumor growth between NAT completion and surgery. The analysis included 106 BC patients who received NAT (69% chemotherapy alone, 31% chemotherapy plus anti-HER2 therapy), had post-NAT breast MRI, and definitive surgery between 2012 and 2019. The median time interval between end-of-treatment and surgery was 6 weeks; 90% had surgery within 8 weeks of NAT completion, and 10% had surgery 8-12 weeks after NAT completion. No significant correlation was found between any of the time intervals (i.e., NAT completion-to-surgery, NAT completion-to-MRI, post-NAT MRI to surgery) and the tumor size as captured in the pathology report. The only parameter that was significantly correlated with pathological tumor size was tumor size as measured on the post NAT MRI (P < .0001). The difference in tumor size between post NAT MRI and surgical pathology did not correlate with the time interval between end-of-treatment and surgery. The ratio between residual tumor size on post-NAT MRI and the time interval from the end-of-treatment to surgery, significantly correlated with the tumor size on surgical pathology (P < .0001) suggesting that NAT has a beneficial effect weeks after end-of-treatment. In conclusion, our results suggest that for patients undergoing neoadjuvant chemotherapy, surgery within 4-8 weeks of completing NAT is reasonable, and is unlikely to result in a clinically significant change in residual tumor size., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Iron-sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression., Competing Interests: The authors declare no conflict of interest.

Maintaining iron (Fe) ion and reactive oxygen species homeostasis is essential for cellular function, mitochondrial integrity and the regulation of cell death pathways, and is recognized as a key process underlying the molecular basis of aging and various diseases, such as diabetes, neurodegenerative diseases and cancer. Nutrient-deprivation autophagy factor 1 (NAF-1; also known as CISD2) belongs to a newly discovered class of Fe-sulfur proteins that are localized to the outer mitochondrial membrane and the endoplasmic reticulum. It has been implicated in regulating homeostasis of Fe ions, as well as the activation of autophagy through interaction with BCL-2. Here we show that small hairpin (sh)RNA-mediated suppression of NAF-1 results in the activation of apoptosis in epithelial breast cancer cells and xenograft tumors. Suppression of NAF-1 resulted in increased uptake of Fe ions into cells, a metabolic shift that rendered cells more susceptible to a glycolysis inhibitor, and the activation of cellular stress pathways that are associated with HIF1α. Our studies suggest that NAF-1 is a major player in the metabolic regulation of breast cancer cells through its effects on cellular Fe ion distribution, mitochondrial metabolism and the induction of apoptosis., (© 2016. Published by The Company of Biologists Ltd.)

Identification of novel drug targets and chemotherapeutic agents is a high priority in the fight against cancer. Here, we report that MAD-28, a designed cluvenone (CLV) derivative, binds to and destabilizes two members of a unique class of mitochondrial and endoplasmic reticulum (ER) 2Fe-2S proteins, mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1), recently implicated in cancer cell proliferation. Docking analysis of MAD-28 to mNT/NAF-1 revealed that in contrast to CLV, which formed a hydrogen bond network that stabilized the 2Fe-2S clusters of these proteins, MAD-28 broke the coordinative bond between the His ligand and the cluster's Fe of mNT/NAF-1. Analysis of MAD-28 performed with control (Michigan Cancer Foundation; MCF-10A) and malignant (M.D. Anderson-metastatic breast; MDA-MB-231 or MCF-7) human epithelial breast cells revealed that MAD-28 had a high specificity in the selective killing of cancer cells, without any apparent effects on normal breast cells. MAD-28 was found to target the mitochondria of cancer cells and displayed a surprising similarity in its effects to the effects of mNT/NAF-1 shRNA suppression in cancer cells, causing a decrease in respiration and mitochondrial membrane potential, as well as an increase in mitochondrial iron content and glycolysis. As expected, if the NEET proteins are targets of MAD-28, cancer cells with suppressed levels of NAF-1 or mNT were less susceptible to the drug. Taken together, our results suggest that NEET proteins are a novel class of drug targets in the chemotherapeutic treatment of breast cancer, and that MAD-28 can now be used as a template for rational drug design for NEET Fe-S cluster-destabilizing anticancer drugs.

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy.

There is an increasing demand for natural compounds that improve women's health by mimicking the critical benefits of estrogen to the bones and the cardiovascular system but avoiding its deleterious effects on the breast and uterus. The estrogenic properties of glabridin, the major isoflavan in licorice root, were tested in view of the resemblance of its structure and lipophilicity to those of estradiol. The results indicate that glabridin is a phytoestrogen, binding to the human estrogen receptor and stimulating creatine kinase activity in rat uterus, epiphyseal cartilage, diaphyseal bone, aorta, and left ventricle of the heart. The stimulatory effects of 2.5-25 microg/animal glabridin were similar to those of 5 microg/animal estradiol. Chemical modification of glabridin showed that the position of the hydroxyl groups has a significant role in binding to the human estrogen receptor and in proliferation-inducing activity. Glabridin was found to be three to four times more active than 2'-O-methylglabridin and 4'-O-methylglabridin, and both derivatives were more active than 2',4'-O-methylglabridin. The effect of increasing concentrations of glabridin on the growth of breast tumor cells was biphasic. Glabridin showed an estrogen receptor-dependent, growth-promoting effect at low concentrations (10 nM-10 microM) and estrogen receptor-independent antiproliferative activity at concentrations of > 15 microM. This is the first study to indicate that isoflavans have estrogen-like activities. Glabridin and its derivatives exhibited varying degrees of estrogen receptor agonism in different tests and demonstrated growth-inhibitory actions on breast cancer cells.

alpha 1-Antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) are closely related protease inhibitors, synthesized primarily by the liver, which play major roles in modulation of the inflammatory response. Previously, we had shown that MCF-7 human breast cancer cells were able to synthesize active alpha 1-AT and alpha 1-ACHY and that the synthesis of both inhibitors varied among different MCF-7 sublines. We now show that when MCF-7(ML) cells (a subline synthesizing low levels of alpha 1-AT) are grown in soft agar in medium depleted of its trypsin inhibitory capacity (i.e. alpha 1-AT-free), addition of alpha 1-AT (50 micrograms/ml) significantly reduces colony formation in both the presence and absence of estradiol (34% and 44%, respectively). Under these conditions, incubation with 10(-7) M estradiol alone increased colony formation 2- to 3-fold. Colony formation was also significantly reduced by serum leukocyte protease inhibitor, which, like alpha 1-AT, is a potent inhibitor of elastase-like enzymes. We also found that a variety of inflammatory mediators, cytokines, and steroid hormones are able to stimulate synthesis of alpha 1-AT and alpha 1-ACHY by MCF-7 cells. Stimulation by interleukin-6 (IL-6; 200 U/ml), epidermal growth factor (4 nM), and estradiol (10(-7) M) was 2- to 3-fold, whereas stimulations by tetradecanoylphorbol-13-acetate (TPA; 80 nM) and IL-1 (10 U/ml) were 2- to 5-fold and 5- to 10-fold, respectively. In each instance, protein synthesis, monitored by immunoprecipitation of 35S-labeled proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady state mRNA levels, monitored by Northern blot analysis with specific cDNA probes, increased to the same extent. Consistent with their ability to stimulate alpha 1-AT synthesis, TPA and IL-1 reduced colony formation in the absence of estradiol by 65% and 63%, respectively. In addition, the effects of both TPA and IL-1 could be reversed by antibody to alpha 1-AT. These results suggest that local synthesis of alpha 1-AT and possibly other protease inhibitors may be important in regulating the tumorigenic potential of MCF-7 breast cancer cells.

We have examined the synthesis of the protease inhibitors alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) by variants of the MCF-7 human breast cancer cell line. Spent medium from MCF-7 203P cells, grown in the absence of serum, was found to contain immunoreactive alpha 1-AT and alpha 1-ACHY by Western blotting. In the presence of 10(-8) M estradiol, levels of both inhibitors were increased 3- to 6-fold. Incubation of spent medium with [125I]trypsin or [125I]chymotrypsin resulted in the formation of stable 75- and 90-kDa complexes identical to the complexes formed between these proteases and the protease inhibitors in plasma, showing the release of active protease inhibitors by MCF-7 cells in culture. Immunoprecipitation of 35S-labeled proteins from the medium of cells grown in the presence of [35S]methionine yielded comparable results, confirming hormonally sensitive synthesis of both protease inhibitors. Northern blot analysis suggests that stimulation of estradiol occurs at the level of transcription. Tetradecanoyl phorbol acetate (50 ng/ml) also stimulated alpha 1-AT and alpha 1-ACHY synthesis 2- to 4-fold, suggesting the involvement of protein kinase-C. Comparison studies with MCF-7 cell sublines ML, BK, 203P, and 300P (a variant spontaneously appearing after 100 passages of 203P) show a wide variation in synthesis of alpha 1-AT and alpha 1-ACHY proteins; sublines 203P and 300P synthesize both inhibitors, the ML subline synthesizes detectable amounts only of alpha 1-ACHY, while no detectable synthesis of either inhibitor was seen in the BK subline. Similar results were obtained for protease inhibitor mRNA transcription by Northern blotting, although low levels of alpha 1-AT mRNA transcription by the ML subline and of alpha 1-AT and alpha 1-ACHY mRNA transcription by the BK subline could be detected.