No abstract

Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of immunocytochemistry, single cell biosensors, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the leading edge. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression at the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.

Breast cancer (BC)‐related mortality primarily results from metastatic colonization of disseminated cells. Actin polymerization plays an important role in driving post‐extravasation metastatic outgrowth of tumor cells. This study examines the role of myocardin‐related transcription factor (MRTF)/serum‐response (SRF), a transcription system well known for regulation of cytoskeletal genes, in metastatic colonization of BC cells. We demonstrated that co‐depletion of MRTF isoforms (MRTF‐A and MRTF‐B) dramatically impairs single‐cell outgrowth ability of BC cells as well as retards growth progression of pre‐established BC cell colonies in three‐dimensional (3D) cultures. Conversely, overexpression of MRTF‐A promotes initiation and progression of tumor‐cell outgrowth in vitro , primary tumor formation, and metastatic outgrowth of seeded BC cells in vivo , and these changes can be dramatically blocked by molecular disruption of MRTF‐A's interaction with SRF. Correlated with the outgrowth phenotypes, we further demonstrate MRTF's ability to augment the intrinsic cellular ability to polymerize actin and formation of F‐actin‐based protrusive structures requiring SRF's interaction. Pharmacological proof‐of‐concept studies show that small molecules capable of interfering with MRTF/SRF signaling robustly suppresses single‐cell outgrowth and progression of pre‐established outgrowth of BC cells in vitro as well as experimental metastatic burden of BC cells in vivo. Based on these data, we conclude that MRTF activity potentiates metastatic colonization of BC cells and therefore, targeting MRTF may be a promising strategy to diminish metastatic burden in BC.

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging.

Poor reproductive performance of dairy cattle has a major negative impact on farm profitability. This is in part attributed to the use of semen from bulls of below-average fertility and to early embryonic mortality. Osteopontin (OPN) is an acidic glycoprotein that has been detected in the epithelium of the ampulla, seminal vesicles, and seminal fluid of high-fertility Holstein bulls. The objective of this study was to determine whether freezing semen with purified bovine milk OPN would improve fertilization and embryonic development in vitro. In a first step, frozen semen from 6 bulls was used with different concentrations of OPN (0, 1, 10, and 100 �g mL-1) to evaluate fertilization, and in a second step, semen from 2 of the 6 bulls was frozen with OPN (0, 5, 10, 20, 40, and 80 �g mL-1) to evaluate its effect on in vitro fertilization and embryonic development. In vitro-matured bovine oocytes were inseminated with 1 � 105 frozen–thawed spermatozoa per 10 oocytes. After 18 h (39�C, 5% CO2 in air), oocytes designated for evaluation of fertilization were fixed, stained, and observed to determine the presence of pronuclei. Oocytes destined for embryo culture were placed in 4-well dishes containing 500 �L of synthetic oviduct fluid per well at 5% O2, 5% CO2, and 90% N2 (v/v). Each experiment was repeated 4 times, and data from each experiment were pooled. Analysis of variance using a general linear model was performed using a weighted mean based on the number of oocytes per treatment. Significantly more (P < 0.05) oocytes were fertilized when frozen semen was used with 10 �g mL-1 of OPN (85 � 4.0%, 78 � 4.0%) from 2 of the 6 bulls than when 0 �g mL-1 was used (75 � 4.0%, 69 � 4.0%). Semen from those bulls resultewd in more fertilized oocytes when 20 �g mL-1 (86 � 3.5%, 79 � 3.4%) and 40 �g mL-1 (88 � 3.4%, 81 � 3.9%) of OPN were used during the second phase of the experiment. Oocytes inseminated with 10 �g mL-1 (84 � 2.5%, 77 � 2.3%), 20 �g mL-1 (87 � 2.8%, 79 � 1.9%), or 40 �g mL-1 (89 � 1.9%, 81 � 2.4%) of OPN in the frozen semen had increased cleavage rates at Day 4 compared with those inseminated with 0 �g mL-1 (75 � 3.5%, 69 � 3.4%). At Day 8, blastocyst development was greater for 10 �g mL-1 (40 � 1.8%, 37 � 1.6%), 20 �g mL-1 (42 � 2.1%, 38 � 2.9%), and 40 �g mL-1 (45 � 2.9%, 40 � 2.5%) of OPN than for the semen with 0 �g mL-1 (33 � 2.3%, 29 � 2.8%). We conclude that semen from some bulls that was frozen with purified bovine milk OPN increases IVF, cleavage, and embryonic development, suggesting a facilitative role for OPN in some reproductive technologies.