Comprehensive Review of Molecular Mechanisms and Clinical Features of Invasive Lobular Cancer.

Invasive lobular carcinoma (ILC) accounts for 10% to 15% of breast cancers in the United States, 80% of which are estrogen receptor (ER)-positive, with an unusual metastatic pattern of spread to sites such as the serosa, meninges, and ovaries, among others. Lobular cancer presents significant challenges in detection and clinical management given its multifocality and multicentricity at presentation. Despite the unique features of ILC, it is often lumped with hormone receptor-positive invasive ductal cancers (IDC); consequently, ILC screening, treatment, and follow-up strategies are largely based on data from IDC. Despite both being treated as ER-positive breast cancer, querying the Cancer Genome Atlas database shows distinctive molecular aberrations in ILC compared with IDC, such as E-cadherin loss (66% vs. 3%), FOXA1 mutations (7% vs. 2%), and GATA3 mutations (5% vs. 20%). Moreover, compared with patients with IDC, patients with ILC are less likely to undergo breast-conserving surgery, with lower rates of complete response following therapy as these tumors are less chemosensitive. Taken together, this suggests that ILC is biologically distinct, which may influence tumorigenesis and therapeutic strategies. Long-term survival and clinical outcomes in patients with ILC are worse than in stage- and grade-matched patients with IDC; therefore, nuanced criteria are needed to better define treatment goals and protocols tailored to ILC's unique biology. This comprehensive review highlights the histologic and clinicopathologic features that distinguish ILC from IDC, with an in-depth discussion of ILC's molecular alterations and biomarkers, clinical trials and treatment strategies, and future targets for therapy. IMPLICATIONS FOR PRACTICE: The majority of invasive lobular breast cancers (ILCs) are hormone receptor (HR)-positive and low grade. Clinically, ILC is treated similar to HR-positive invasive ductal cancer (IDC). However, ILC differs distinctly from IDC in its clinicopathologic characteristics and molecular alterations. ILC also differs in response to systemic therapy, with studies showing ILC as less sensitive to chemotherapy. Patients with ILC have worse clinical outcomes with late recurrences. Despite these differences, clinical trials treat HR-positive breast cancers as a single disease, and there is an unmet need for studies addressing the unique challenges faced by patients diagnosed with ILC.

A Bias in the Reading of the Genetic Code of Escherichia coli is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins.

BACKGROUND: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. MATERIALS AND METHODS: Based on the data from the EcoCyc website of the National Center for Biotechnology Information (NCBI), the sequence of all E. coli MG1655 genes were scanned for ACA sites upstream from the initiation codons. Among these sequences, the fuzznuc program of the "European Molecular Biology Open Software Suite" (EMBOSS) was used to find the "ACA" pattern. The distribution of the ACA threonine codon, both in-frame and out-of-frame, was determined by using the HTML Script Program (Supplementary Material). RESULTS: Here it is reported that for most of the E. coli proteins mediated by stress-induced MazF, the ACA threonine codon in their mRNAs is not in-frame but rather out-of-frame; in these same RNAs, the three synonymous threonine codons, ACG, ACU, and ACC, are in-frame. In contrast, for proteins translated by the canonical translation system, in the majority of mRNAs, the ACA codon is located in-frame. CONCLUSION: The described bias in the genetic code is a characteristic of E. coli genes specifying for stress-induced MazF-mediated proteins.

Stress-Induced MazF-Mediated Proteins in Escherichia coli.

Escherichia colimazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions.IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

Quorum Sensing Extracellular Death Peptides Enhance the Endoribonucleolytic Activities of Mycobacterium tuberculosis MazF Toxins.

mazEF is a toxin-antitoxin module located on chromosomes of most bacteria. MazF toxins are endoribonucleases antagonized by MazE antitoxins. Previously, we characterized several quorum sensing peptides called "extracellular death factors" (EDFs). When secreted from bacterial cultures, EDFs induce interspecies cell death. EDFs also enhance the endoribonucleolytic activity of Escherichia coli MazF. Mycobacterium tuberculosis carries several mazEF modules. Among them, the endoribonucleolytic activities of MazF proteins mt-1, mt-3, and mt-6 were identified. MazF-mt6 and MazF-mt-3 cleave M. tuberculosis rRNAs. Here we report the in vitro effects of EDFs on the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3 and overcome the inhibitory effect of MazE-mt3 or MazE-mt6 on the endoribonucleolytic activities of the respective toxins. We propose that these EDFs can serve as a basis for a novel class of antibiotics against M. tuberculosisIMPORTANCEMycobacterium tuberculosis is one of the leading causes of death from infectious disease. M. tuberculosis is highly drug resistant, and drug delivery to the infected site is very difficult. In previous studies, we showed that extracellular death factors (EDFs) can work as quorum sensing molecules which participate in interspecies bacterial cell death. In this study, we demonstrated the role of different EDFs in the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3. The current report provides a basis for the use of the EDF peptides EcEDF and PaEDF as novel antibiotics against M. tuberculosis.

Cytogenomics of hexavalent chromium (Cr 6+) exposed cells: a comprehensive review.

The altered cellular gene expression profile is being hypothesized as the possible molecular basis navigating the onset or progress of various morbidities. This hypothesis has been evaluated here in respect of Cr 6+ induced toxicity. Several studies using gene microarray show selective and strategic dysregulations of cellular genes and pathways induced by Cr 6+. Relevant literature has been reviewed to unravel these changes in different test systems after exposure to Cr 6+ and also to elucidate association if any, of the altered cytogenomics with Cr 6+ induced toxicity or carcinogenicity. The aim was to verify the hypothesis for critical role of altered cytogenomics in onset of Cr 6+ induced biological/clinical effects by identifying genes modulated commonly by the toxicant irrespective of test system or test concentrations/doses, and by scrutinizing their importance in regulation of the flow of mechanistically linked events crucial for resultant morbidities. Their probability as biomarkers to monitor the toxicant induced biological changes is speculative. The modulated genes have been found to cluster under the pathways that manage onset of oxidative stress, DNA damage, apoptosis, cell-cycle regulation, cytoskeleton, morphological changes, energy metabolism, biosynthesis, oncogenes, bioenergetics, and immune system critical for toxicity. In these studies, the identity of genes has been found to differ remarkably; albeit the trend of pathways' dysregulation has been found to remain similar. We conclude that the intensity of dysregulation of genes or pathways involved in mechanistic events forms a sub-threshold or threshold level depending upon the dose and type (including speciation) of the toxicant, duration of exposure, type of target cells, and niche microenvironment of cells, and the intensity of sub-threshold or threshold level of the altered cytogenomics paves way in toxicant exposed cells eventually either to opt for reversal to differentiation and growth, or to result in toxicity like dedifferentiation and apoptosis, respectively.

Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways.

Murine or human cancer cells have high glutathione levels. Depletion of the elevated GSH inhibits proliferation of cancer cells. Molecular basis for this observation is little understood. In an attempt to find out the underlying mechanism, we reproduced these effects in transformed C3H10T1/2 and BALB/c 3T3 cells using diethyl maleate and studied cytogenomic changes in the whole mouse genome using spotted 8 × 60 K arrays. Transformed cells revealed an increase in GSH levels. GSH depletion by DEM inhibited the growth of transformed cells. The non-cytotoxic dose of DEM (0.25 mM) resulted in GSH depletion, ROS generation, cell cycle arrest, apoptosis, decrease in anchorage independent growth, gene expression changes and activation of all three members of the MAPK family. Increase in intracellular GSH levels by GSHe countered the effect of DEM. These results support the physiological importance of GSH in regulation of gene expression for transformed cell growth restraint. This study is of interest in not only understanding the molecular biology of the transformed cells, but also in identifying new targets for development of gene therapy together with the chemotherapy.

Lipoic acid prevents Cr(6+) induced cell transformation and the associated genomic dysregulation.

Investigation of the transcription profile of cells transformed by Cr(6+) in vivo was undertaken. The objective was to elucidate genomic changes underlying the mechanism of action of the carcinogenic dose of Cr(6+)and their prevention using metabolic antioxidant lipoic acid (LA). Cr(6+) was administered intraperitoneally to LPS+TPA challenged Swiss albino mice in host mediated cell transformation assay using peritoneal macrophages in vivo. The cell transforming potential of Cr(6+) test doses was validated by gain of anchorage independent growth potential in soft agar and loss of Fc receptor on target cells. LA was administered in equimolar doses. Compared to non-transformed cells, the gene expression profile of transformed cells was found to be dysregulated substantially and in dose dependent manner. Genes showing down regulation were found to be involved in tumour suppression, apoptosis, DNA repair, and cell-cycle. A similar response was noted in the genes pertaining to immune system, morphogenesis, cell-communication, energy-metabolism, and biosynthesis. The co-administration of lipoic acid prevented the transcription dysregulation and cell transformation by Cr(6+) in vivo. The influenced pathways seem to be crucial for progression as well as mitigation of Cr toxicity; and their response to LA indicated their critical role in mechanism of anti-carcinogenic action of LA. Results are of importance to mitigate Cr(6+) induced occupational cancer hazard.

Dysregulation of pathways involved in the processing of cancer and microenvironment information in MCA + TPA transformed C3H/10T1/2 cells.

The two-stage cell transformation assay is an in vitro model cell culture system to identify the ability of chemicals to act as initiators or promoters of cell transformation and also to study the cellular and molecular mechanisms of chemically induced morphological and neoplastic cell transformation. The global gene expression profiles of 3-methylcholanthrene (MCA) + 12-O-tetradecanoylphorbol-13-acetate (TPA)-transformed C3H/10T1/2 cells are not known. Therefore, we have investigated the global transcriptional profile of MCA + TPA-transformed C3H10T1/2 cells using an 8 × 60 k probe microarray. The study revealed a differential regulation of pathways and gene expressions. Multifold dysregulation was seen in pathways of cancer, phagosomal activity, and tumor cell microenvironment information processing systems, notably the neuroactive ligand-receptor interaction, actin cytoskeleton regulation, tight junction, axon guidance, and cell adhesion molecules. The genes FGF1, EIF4E1B, MAGI1, and GRIA3 showed upregulation; these encoded the pluripotent fibroblast growth factor, the translation initiation factor, the tight junction scaffolding protein, and the antiapoptotic as well as the enhancer of proliferation and migration, respectively. The genes CXCL7/CXCL5/CXCL12, H2DMB1, and HSPA1A showed downregulation; these encoded the chemotactic agent protein, the protein involved in MHC class II antigen processing/presentation or participating in cell adhesion/phagosomal activity/autoimmune disorder, and the chaperone protein stabilizing the existing as well as newly translated cytosolic/organelle proteins against aggregation, respectively. By loss or gain of function, these dysregulated genes apparently seem to reprogram cells for apoptosis or proliferation and support their transformation into the tumor cell phenotype. The observed molecular changes can be seen as molecular signatures of transformed cells and can be of use as objective evidences to C3H/10T1/2 cell transformation assay in investigations on the carcinogenic potential of chemicals and their mechanism of actions using in vitro carcinogenesis method.

Cytoprotection against Cr(6+)-induced DNA damage by alpha-lipoic acid: implications in reducing occupational cancer risk.

Alpha-lipoic acid (LA), the metabolic antioxidant, was evaluated for its potential to protect against Cr(6+)-induced DNA damage. Potassium dichromate was administered to Swiss albino mice orally ad libitum at the doses of 5, 10 or 25 mg/kg body weight in drinking water to set DNA damage in cells, which was characterized in mouse peripheral blood mononuclear cells and bone marrow cells using single-cell gel electrophoresis and analyses of generated comets for Tail moment, Tail DNA and Tail length. DNA damage was dose dependent. Cytoprotection by LA was remarkable. LA (5, 10 and 25 mg/kg body weight intraperitoneally) in pre-, co- and post-toxicant administration schedule abrogated DNA damage substantially in both cell types. Protection by LA was also dose dependent. LA annulled DNA damage by Cr(6+) in plasmid relaxation assay. A negligible DNA damage resulted during interaction of Cr(6+) and LA. Compared to ascorbate, LA emerged as a better antioxidant and least DNA damaging. In conclusion, our study advocated an experimental therapeutic research potential in LA against Cr(6+)-induced DNA damage for reduction of occupational cancer risk in humans.