Genome folding principles uncovered in condensin-depleted mitotic chromosomes.

During mitosis, condensin activity is thought to interfere with interphase chromatin structures. To investigate genome folding principles in the absence of chromatin loop extrusion, we codepleted condensin I and condensin II, which triggered mitotic chromosome compartmentalization in ways similar to that in interphase. However, two distinct euchromatic compartments, indistinguishable in interphase, emerged upon condensin loss with different interaction preferences and dependencies on H3K27ac. Constitutive heterochromatin gradually self-aggregated and cocompartmentalized with facultative heterochromatin, contrasting with their separation during interphase. Notably, some cis-regulatory element contacts became apparent even in the absence of CTCF/cohesin-mediated structures. Heterochromatin protein 1 (HP1) proteins, which are thought to partition constitutive heterochromatin, were absent from mitotic chromosomes, suggesting, surprisingly, that constitutive heterochromatin can self-aggregate without HP1. Indeed, in cells traversing from M to G1 phase in the combined absence of HP1α, HP1ß and HP1γ, constitutive heterochromatin compartments are normally re-established. In sum, condensin-deficient mitotic chromosomes illuminate forces of genome compartmentalization not identified in interphase cells.

Robotic Versus Laparoscopic Surgery for Rectal Cancer: A Comprehensive Review of Oncological Outcomes.

The treatment of rectal cancer is complex and involves specialized multidisciplinary care, although the tenet is still rooted in a high-quality total mesorectal excision. The robotic platform is one of many tools in the arsenal to assist dissection in the low pelvis. This article is a comprehensive review of the oncological outcome comparing robotic vs laparoscopic rectal cancer resection, with a particular focus on total mesorectal excision. There is no statistical difference in total mesorectal grade, circumferential margin, distal margin, and lymph node harvest. Survival data are less mature, but there is also no difference in disease-free or overall survival between the two techniques. Although additional randomized trials are still needed to validate these findings, both techniques are currently acceptable in the minimally invasive treatment of rectal cancer, and surgeon preference is paramount to safe and optimal resection.

LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

SMAD pathways govern epithelial proliferation, and transforming growth factor ß (TGF-ß and BMP signaling through SMAD members has distinct effects on mammary development and homeostasis. Here, we show that LEFTY1, a secreted inhibitor of NODAL/SMAD2 signaling, is produced by mammary progenitor cells and, concomitantly, suppresses SMAD2 and SMAD5 signaling to promote long-term proliferation of normal and malignant mammary epithelial cells. In contrast, BMP7, a NODAL antagonist with context-dependent functions, is produced by basal cells and restrains progenitor cell proliferation. In normal mouse epithelium, LEFTY1 expression in a subset of luminal cells and rare basal cells opposes BMP7 to promote ductal branching. LEFTY1 binds BMPR2 to suppress BMP7-induced activation of SMAD5, and this LEFTY1-BMPR2 interaction is specific to tumor-initiating cells in triple-negative breast cancer xenografts that rely on LEFTY1 for growth. These results suggest that LEFTY1 is an endogenous dual-SMAD inhibitor and that suppressing its function may represent a therapeutic vulnerability in breast cancer.

Methylation of High-Risk Human Papillomavirus Genomes Are Associated with Cervical Precancer in HIV-Positive Women.

BACKGROUND: HIV-positive women are at substantial risk of HPV-associated cervical neoplasia caused by high-risk (HR) HPVs. Methylation of the HPV genome is associated with cervical intraepithelial neoplasia grade 3 (CIN3) in HIV-negative women, yet it is unknown whether this holds true for HIV-positive women. METHODS: We designed a case-control study within the Women's Interagency HIV Study (WIHS) cohort comparing HIV-positive CIN3 cases (N = 72) to HIV-positive controls without detectable CIN2+. The unit of analysis and matching was HPV-type infection. Cases with ≥2 HR-HPV types (N = 23; 32%) had a separate control for each HR-HPV type. We developed and utilized next-generation sequencing (NGS) methylation assays for 12 different HR-HPVs, focusing on CpG sites in the L1/L2 regions. RESULTS: Significant case-control differences in individual CpG site methylation levels were observed for multiple alpha-9 (HPV16/31/35/58) and alpha-7 HPV (HPV18/39/45) types, based on dichotomization of tertile levels (T3 vs. T1 and T2). Analyses combining homologous CpG sites [e.g., HPV16-L1-5608/HPV31-L1-5521/HPV35-L2L1-5570; OR = 7.28; 95% confidence interval (CI): 2.75-19.3], and (e.g., HPV18-L1-7062/HPV45-L1-7066; OR = 6.94; 95% CI: 1.23-39.3) were significant in separate case-control comparisons. In cases with multiple HR-HPVs, we tested and confirmed the hypothesis that one HR-HPV type would have higher methylation than other types detected, consistent with there being a single HR-HPV causally related to a lesion. CONCLUSIONS: CIN3 is associated with elevated L1/L2 CpG methylation levels in HIV-positive women. IMPACT: HPV DNA CpG methylation is a promising triage option in HIV-positive women testing positive for HR-HPV types and provides risk attribution in women with multiple HPV type infections.

End-of-treatment and serial PET imaging in primary mediastinal B-cell lymphoma following dose-adjusted EPOCH-R: a paradigm shift in clinical decision making.

Dose-adjusted-EPOCH-R obviates the need for radiotherapy in most patients with primary mediastinal B-cell lymphoma. End-of-treatment PET, however, does not accurately identify patients at risk of treatment failure, thereby confounding clinical decision making. To define the role of PET in primary mediastinal B-cell lymphoma following dose-adjusted-EPOCH-R, we extended enrollment and follow up on our published phase II trial and independent series. Ninety-three patients received dose-adjusted-EPOCH-R without radiotherapy. End-of-treatment PET was performed in 80 patients, of whom 57 received 144 serial scans. One nuclear medicine physician from each institution blindly reviewed all scans from their respective institution. End-of-treatment PET was negative (Deauville 1-3) in 55 (69%) patients with one treatment failure (8-year event-free and overall survival of 96.0% and 97.7%). Among 25 (31%) patients with a positive (Deauville 4-5) end-of-treatment PET, there were 5 (20%) treatment failures (8-year event-free and overall survival of 71.1% and 84.3%). Linear regression analysis of serial scans showed a significant decrease in SUVmax in positive end-of-treatment PET non-progressors compared to an increase in treatment failures. Among 6 treatment failures, the median end-of-treatment SUVmax was 15.4 (range, 1.9-21.3), and 4 achieved long-term remission with salvage therapy. Virtually all patients with a negative end-of-treatment PET following dose-adjusted-EPOCH-R achieved durable remissions and should not receive radiotherapy. Among patients with a positive end-of-treatment PET, only 5/25 (20%) had treatment-failure. Serial PET imaging distinguished end-of-treatment PET positive patients without treatment failure, thereby reducing unnecessary radiotherapy by 80%, and should be considered in all patients with an initial positive PET following dose-adjusted-EPOCH-R (clinicaltrials.gov identifier 00001337).

Human Papillomavirus DNA Methylation as a Biomarker for Cervical Precancer: Consistency across 12 Genotypes and Potential Impact on Management of HPV-Positive Women.

Purpose: Human papillomavirus (HPV) DNA methylation testing is a promising triage option for women testing HPV positive during cervical cancer screening. However, the extent to which methylation indicates precancer for all 12 carcinogenic HPV types has not been evaluated.Experimental Design: In this nested case-control study, we tested up to 30 cases of precancer [cervical intraepithelial neoplasia grade 3 (CIN3)/adenocarcinoma in situ (AIS)] and 30 normal controls for each carcinogenic type (single infections with 16/18/31/33/35/39/45/51/52/56/58/59). Next-generation bisulfite sequencing was performed on CpG sites within the L1 and L2 genes. We calculated differences in methylation, ORs, and AUC. Using a fixed sensitivity of 80%, we evaluated the specificity and the risk of CIN3/AIS for best performing CpG sites, and compared the performance of an explorative multi-type methylation assay with current triage strategies.Results: Methylation was positively associated with CIN3/AIS across all 12 types. AUCs for the top sites ranged from 0.71 (HPV51 and HPV56) to 0.86 (HPV18). A combined 12-type methylation assay had the highest Youden index (0.46), compared with cytology (0.31) and a 5-type methylation assay, including only previously described types (0.26). The 12-type methylation assay had higher sensitivity (80% vs. 76.6%) and lower test positivity compared with cytology (38.5% vs. 48.7%). The risk of CIN3/AIS was highest for methylation positives and lowest for cytology or HPV16/18 positives.Conclusions: HPV DNA methylation is a general phenomenon marking the transition from HPV infection to precancer for all 12 carcinogenic types. Development of a combined multitype methylation assay may serve as a triage test for HPV-positive women. Clin Cancer Res; 24(9); 2194-202. ©2018 AACR.

A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland.

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11bhigh cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G0 phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as a central intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.

High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.

Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle weakness, ultimately leading to early mortality in affected teenagers and young adults. Previous work from our lab has shown that a small transmembrane protein called sarcospan (SSPN) can enhance the recruitment of adhesion complex proteins to the cell surface. When human SSPN is expressed at three-fold levels in mdx mice, this increase in adhesion complex abundance improves muscle membrane stability, preventing many of the histopathological changes associated with DMD. However, expressing higher levels of human SSPN (ten-fold transgenic expression) causes a severe degenerative muscle phenotype in wild-type mice. Since SSPN-mediated stabilization of the sarcolemma represents a promising therapeutic strategy in DMD, it is important to determine whether SSPN can be introduced at high levels without toxicity. Here, we show that mouse SSPN (mSSPN) can be overexpressed at 30-fold levels in wild-type mice with no deleterious effects. In mdx mice, mSSPN overexpression improves dystrophic pathology and sarcolemmal stability. We show that these mice exhibit increased resistance to eccentric contraction-induced damage and reduced fatigue following exercise. mSSPN overexpression improved pulmonary function and reduced dystrophic histopathology in the diaphragm. Together, these results demonstrate that SSPN overexpression is well tolerated in mdx mice and improves sarcolemma defects that underlie skeletal muscle and pulmonary dysfunction in DMD.

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition.

BACKGROUND: The BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes. METHODS: SW13 subtype morphology, chemotaxis, growth rates, and gene expression were assessed by standard immunofluorescence, transwell, growth, and qPCR assays. Metastatic potential was measured by anchorage-independent growth and MMP activity. The efficacy of HDAC inhibitors in inducing subtype switching was determined by immunofluorescence and qPCR. Histone modifications were assessed by western blot. RESULTS: Treatment of SW13- cells with HDAC1 inhibitors most effectively promotes re-expression of BRM and VIM, characteristic of the SW13+ phenotype. During treatment, hyperacetylation of histone residues and hypertrimethylation of H3K4 is pronounced. Furthermore, histone modification enzymes, including HDACs and KDM5C, are differentially expressed during treatment but several features of this differential expression pattern differs from that seen in the SW13- and SW13+ subtypes. As the SW13- subtype is more proliferative while the SW13+ subtype is more metastatic, treatment with HDACi increases the metastatic potential of SW13 cells while restoring expression of the BRM tumor suppressor. CONCLUSIONS: When compared to the SW13- subtype, SW13+ cells have restored BRM expression, increased metastatic capacity, and significantly different expression of a variety of chromatin remodeling factors including those involved with histone acetylation and methylation. These data are consistent with a multistep mechanism of SW13- to SW13+ conversion and subtype stabilization: histone hypermodification results in the altered expression of chromatin remodeling factors and chromatin epigenetic enzymes and the re-expression of BRM which results in restoration of SWI/SNF complex function and leads to changes in chromatin structure and gene expression that stabilize the SW13+ phenotype.

Accuracy of MR imaging-estimated proton density fat fraction for classification of dichotomized histologic steatosis grades in nonalcoholic fatty liver disease.

PURPOSE: To evaluate the diagnostic performance of previously proposed high-specificity magnetic resonance (MR) imaging-estimated proton density fat fraction (PDFF) thresholds for diagnosis of steatosis grade 1 or higher (PDFF threshold of 6.4%), grade 2 or higher (PDFF threshold of 17.4%), and grade 3 (PDFF threshold of 22.1%) by using histologic findings as a reference in an independent cohort of adults known to have or suspected of having nonalcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: This prospective, cross-sectional, institutional review board-approved, HIPAA-compliant single-center study was conducted in an independent cohort of 89 adults known to have or suspected of having NAFLD who underwent contemporaneous liver biopsy. MR imaging PDFF was estimated at 3 T by using magnitude-based low-flip-angle multiecho gradient-recalled-echo imaging with T2* correction and multipeak modeling. Steatosis was graded histologically (grades 0, 1, 2, and 3, according to the Nonalcoholic Steatohepatitis Clinical Research Network scoring system). Sensitivity, specificity, and binomial confidence intervals were calculated for the proposed MR imaging PDFF thresholds. RESULTS: The proposed MR imaging PDFF threshold of 6.4% to diagnose grade 1 or higher steatosis had 86% sensitivity (71 of 83 patients; 95% confidence interval [CI]: 76, 92) and 83% specificity (five of six patients; 95% CI: 36, 100). The threshold of 17.4% to diagnose grade 2 or higher steatosis had 64% sensitivity (28 of 44 patients; 95% CI: 48, 78) and 96% specificity (43 of 45 patients; 95% CI: 85, 100). The threshold of 22.1% to diagnose grade 3 steatosis had 71% sensitivity (10 of 14 patients; 95% CI: 42, 92) and 92% specificity (69 of 75 patients; 95% CI: 83, 97). CONCLUSION: In an independent cohort of adults known to have or suspected of having NAFLD, the previously proposed MR imaging PDFF thresholds provided moderate to high sensitivity and high specificity for diagnosis of grade 1 or higher, grade 2 or higher, and grade 3 steatosis. Prospective multicenter studies are now needed to further validate these high-specificity thresholds.

miR-142 regulates the tumorigenicity of human breast cancer stem cells through the canonical WNT signaling pathway.

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression.

A cell-intrinsic role for TLR2-MYD88 in intestinal and breast epithelia and oncogenesis.

It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.

The fate of the primary cilium during myofibroblast transition.

Myofibroblasts, the culprit of organ fibrosis, can originate from mesenchymal and epithelial precursors through fibroblast-myofibroblast and epithelial-myofibroblast transition (EMyT). Because certain ciliopathies are associated with fibrogenesis, we sought to explore the fate and potential role of the primary cilium during myofibroblast formation. Here we show that myofibroblast transition from either precursor results in the loss of the primary cilium. During EMyT, initial cilium growth is followed by complete deciliation. Both EMyT and cilium loss require two-hit conditions: disassembly/absence of intercellular contacts and transforming growth factor-ß1 (TGFß) exposure. Loss of E-cadherin-dependent junctions induces cilium elongation, whereas both stimuli are needed for deciliation. Accordingly, in a scratch-wounded epithelium, TGFß provokes cilium loss exclusively along the wound edge. Increased contractility, a key myofibroblast feature, is necessary and sufficient for deciliation, since constitutively active RhoA, Rac1, or myosin triggers, and down-regulation of myosin or myocardin-related transcription factor prevents, this process. Sustained myosin phosphorylation and consequent deciliation are mediated by a Smad3-, Rac1-, and reactive oxygen species-dependent process. Transitioned myofibroblasts exhibit impaired responsiveness to platelet-derived growth factor-AA and sonic hedgehog, two cilium-associated stimuli. Although the cilium is lost during EMyT, its initial presence contributes to the transition. Thus myofibroblasts represent a unique cilium-less entity with profoundly reprogrammed cilium-related signaling.

Utility of magnetic resonance imaging versus histology for quantifying changes in liver fat in nonalcoholic fatty liver disease trials.

UNLABELLED: The magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF) is a novel imaging-based biomarker that allows fat mapping of the entire liver, whereas the magnetic resonance spectroscopy-measured proton density fat fraction (MRS-PDFF) provides a biochemical measure of liver fat in small regions of interest. Cross-sectional studies have shown that MRI-PDFF correlates with MRS-PDFF. The aim of this study was to show the utility of MRI-PDFF in assessing quantitative changes in liver fat through a three-way comparison of MRI-PDFF and MRS-PDFF with the liver histology-determined steatosis grade at two time points in patients with nonalcoholic fatty liver disease (NAFLD). Fifty patients with biopsy-proven NAFLD who participated in a randomized trial underwent a paired evaluation with liver biopsy, MRI-PDFF, and MRS-PDFF at the baseline and 24 weeks. The mean age and body mass index were 47.8 ± 11.7 years and 30.7 ± 6.5 kg/m(2), respectively. MRI-PDFF showed a robust correlation with MRS-PDFF both at week 0 and at week 24 (r = 0.98, P < 0.0001 for both). Cross-sectionally, MRI-PDFF and MRS-PDFF increased with increases in the histology-determined steatosis grade both at week 0 and at week 24 (P < 0.05 for all). Longitudinally, patients who had a decrease (≥ 1%) or increase (≥ 1%) in MRI-PDFF (confirmed by MRS-PDFF) showed a parallel decrease or increase in their body weight and serum alanine aminotransferase and aspartate aminotransferase levels at week 24 (P < 0.05). This small increase or decrease in liver fat could not be quantified with histology. CONCLUSION: In this longitudinal study, MRI-PDFF correlated well with MRS-PDFF and was more sensitive than the histology-determined steatosis grade in quantifying increases or decreases in the liver fat content. Therefore, it could be used to quantify changes in liver fat in future clinical trials.

Remodeling of endogenous mammary epithelium by breast cancer stem cells.

Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC.

Association of reactive oxygen species levels and radioresistance in cancer stem cells.

The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.