Metastasis is an Early Event in Mouse Mammary Carcinomas and is Associated with Cells Bearing Stem Cell Markers

Introduction: It is still uncertain whether metastasis is predominantly an early or late event in tumor progression. The detection of early metastases and cells responsible for the dissemination may therefore have significant clinical implications. Methods: Lung dissemination and/or metastasis were investigated in mice carrying the polyomavirus middle-T oncogene (PyMT) during different stages of mammary tumorigenesis using the colony forming assay. Immunocytochemical or immunohistochemical staining was used to identify subpopulations of cells responsible for lung dissemination and metastasis. Histological examination was used to show primary and metastatic tumors. The tumor-initiating and metastatic capacity of cells expressing stem cell markers was assessed in syngeneic wild-type (WT) mice whose mammary fat pads were injected with these cells. Results: Metastatic mammary epithelial cells were detected in the lungs of mice carrying the PyMT oncogene (MMT mice). These cells were observed early in breast tumorigenesis when the mammary tree appeared by histological inspection to be normal (or at a premalignant stage), suggesting the possession of disseminating and metastatic capacity even before full malignant transformation. Some of the disseminated cells and lung metastases displayed surface stem cell markers. These findings suggest that stem cells from apparently precancerous primary lesions could be a source of metastasis. Indeed, injection of lung tissue cells from MMT mice into syngeneic WT mice resulted in the formation of mammary tumors. These tumors resembled their parent mammary tumors in the MMT donors as well as grafted tumors derived from mammary tumor cells. Furthermore, when we injected lung tissue cells from GFP MMT mice into the fat pads of recipient WT mice, disseminated or metastatic GFP-expressing cells were detected in the lungs, lymph nodes and blood of the recipient WT mice. We finally identified a subpopulation of mammary epithelial/tumor cells expressing CD44 and Sca1 that was largely responsible for dissemination and metastasis in MMT mice. Conclusions: The tumorigenic and metastatic potential of a subpopulation of mammary epithelial/tumor cells in MMT mice is endowed relatively early in mammary neoplasms and suggests a potential role for cancer stem cell sub-populations in metastasis

Genotoxic stress induces Sca‐1‐expressing metastatic mammary cancer cells

We describe a cell damage‐induced phenotype in mammary carcinoma cells involving acquisition of enhanced migratory and metastatic properties. Induction of this state by radiation required increased activity of the Ptgs2 gene product cyclooxygenase 2 (Cox2), secretion of its bioactive lipid product prostaglandin E2 (PGE2), and the activity of the PGE2 receptor EP4. Although largely transient, decaying to low levels in a few days to a week, this phenotype was cumulative with damage and levels of cell markers Sca‐1 and ALDH1 increased with treatment dose. The Sca‐1+, metastatic phenotype was inhibited by both Cox2 inhibitors and PGE2 receptor antagonists, suggesting novel approaches to radiosensitization

Radiosensitization of mammary carcinoma cells by telomere homolog oligonucleotide pretreatment

Introduction: Ionizing radiation (IR) is a widely used approach to cancer therapy, ranking second only to surgery in rate of utilization. Responses of cancer patients to radiotherapy depend in part on the intrinsic radiosensitivity of the tumor cells. Thus, promoting tumor cell sensitivity to IR could significantly enhance the treatment outcome and quality of life for patients. Methods: Mammary tumor cells were treated by a 16-base phosphodiester-linked oligonucleotide homologous to the telomere G-rich sequence TTAGGG (T-oligo: GGTTAGGTGTAGGTTT) or a control-oligo (the partial complement, TAACCCTAACCCTAAC) followed by IR. The inhibition of tumor cell growth in vitro was assessed by cell counting and clonogenic cell survival assay. The tumorigenesis of tumor cells after various treatments was measured by tumor growth in mice. The mechanism underlying the radiosensitization by T-oligo was explored by immunofluorescent determination of phosphorylated histone H2AX ($\gamma$H2AX) foci, $\beta$-galactosidase staining, comet and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assays. The efficacy of the combined treatment was assessed in a spontaneous murine mammary tumor model. Results: Pretreatment of tumor cells with T-oligo for 24 hours in vitro enhanced both senescence and apoptosis of irradiated tumor cells and reduced clonogenic potential. Radiosensitization by T-oligo was associated with increased formation and/or delayed resolution of $\gamma$H2AX DNA damage foci and fragmented DNA. T-oligo also caused radiosensitization in two in vivo mammary tumor models. Indeed, combined T-oligo and IR-treatment in vivo led to a substantial reduction in tumor growth. Of further significance, treatment with T-oligo and IR led to synergistic inhibition of the growth of spontaneous mammary carcinomas. Despite these profound antitumor properties, T-oligo and IR caused no detectable side effects under our experimental conditions. Conclusions: Pretreatment with T-oligo sensitizes mammary tumor cells to radiation in both in vitro and in vivo settings with minimal or no normal tissue side effects

Overexpression of Soybean <i>GmWRI1a</i> Stably Increases the Seed Oil Content in Soybean

WRINKLED1 (WRI1), an APETALA2/ethylene-responsive-element-binding protein (AP2/EREBP) subfamily transcription factor, plays a crucial role in the transcriptional regulation of plant fatty acid biosynthesis. In this study, GmWRI1a was overexpressed in the soybean cultivar ‘Dongnong 50’ using Agrobacterium-mediated transformation to generate three transgenic lines with high seed oil contents. PCR and Southern blotting analysis showed that the T-DNA was inserted into the genome at precise insertion sites and was stably inherited by the progeny. Expression analysis using qRT-PCR and Western blotting indicated that GmWRI1a and bar driven by the CaMV 35S promoter were significantly upregulated in the transgenic plants at different developmental stages. Transcriptome sequencing results showed there were obvious differences in gene expression between transgenic line and transgenic receptor during seed developmental stages. KEGG analysis found that the differentially expressed genes mainly annotated to metabolic pathways, such as carbohydrated metabolism and lipid metabolism. A 2-year single-location field trial revealed that three transgenic lines overexpressing GmWRI1a (GmWRI1a-OE) showed a stable increase in seed oil content of 4.97–10.35%. Importantly, no significant effect on protein content and yield was observed. Overexpression of GmWRI1a changed the fatty acid composition by increasing the linoleic acid (C18:2) content and decreasing the palmitic acid (C16:0) content in the seed. The three GmWRI1a-OE lines showed no significant changes in agronomic traits. The results demonstrated that the three GmWRI1a overexpression lines exhibited consistent increases in seed oil content compared with that of the wild type and did not significantly affect the seed yield and agronomic traits. The genetic engineering of GmWRI1a will be an effective strategy for the improvement of seed oil content and value in soybean