Whsc1l1 Regulates Estrogen Receptor Activity In Sum44 Breast Cancer Cells

Breast cancer is the most common cancer and the second leading cause of cancer death in women. While ER-positive breast cancer subtypes are initially well-managed by targeted therapies targeting estrogen signaling, many women are suffering from recurrence of a more aggressive, hormone insensitive cancer 5 or more years after initial remission. Late recurrence of hormone resistant breast cancer in patients who were previously successfully treated with anti-estrogen therapies worsens overall long-term outcomes, and specific oncogenic mutations may be driving late recurring aggressive disease in these patients. More complete characterization of the oncogenome of a tumor may allow for the possibility of customized therapy targeting each patient\u27s specific oncogene-activating mutations, and in doing so increase the probability of durable remission over the long term. Basic research on cell lines modeling different breast cancer subtypes makes possible discovery and characterization of novel driving oncogenes and their context in a breast cancer subtype model. The Wolf Hirschhorn Syndrome Candidate 1-Like 1 gene (WHSC1L1) is one of approximately 50 genes in the chromosome 8p11-p12 amplicon, an amplified region of the short arm of chromosome 8 found in 12-15% of human breast cancers, as well as other cancer types such as lung. Amplification of the 8p11-p12 region is most often found in breast cancers of the luminal B subtype. WHSC1L1 is a member of the NSD family of histone lysine methyltransferases, SET domain-containing proteins which catalyze the addition of a methyl group to lysines on the amino-terminal tail of histone H3 subunits. The WHSC1L1 gene expresses two known isoforms which code for two distinct proteins, WHSC1L1-long and WHSC1L1-short. The short isoform of WHSC1L1 codes for the first 647 of the 1437 amino acids present in the long isoform, and lacks the catalytic SET domain and several PHD and PWWP chromatin interacting domains, containing a single PWWP domain and a recently characterized acidic transactivation domain. In both normal and tumor breast tissue, WHSC1L1-short is expressed at greater levels than WHSC1L1-long. Several breast cancer cell lines established in the Ethier lab harbor WHSC1L1 amplifications and also overexpress both isoforms of WHSC1L1. The SUM44 cell line is a highly ER-positive cell line model of luminal B breast cancer isolated from a pleural effusion metastasis of a patient with aggressive disease. It is known that the short isoform of WHSC1L1, WHSC1L1-short, is a potent driving oncogene in SUM44 cells, however the specific mechanism of WHSC1L1-short as an oncomodifier is not known. To investigate WHSC1L1-short function as an oncogene in SUM44 cells, we developed an shRNA knockdown model that specifically knocked down expression of WHSC1L1-short through its unique 3\u27 UTR sequence (shWHSC1L1-short), and a model that knocked down both WHSC1L1 isoforms (shWHSC1L1-total). We found that knockdown of both total WHSC1L1 and WHSC1L1-short alone negatively affected SUM44 proliferation, and that WHSC1L1-short knockdown had a larger effect than knockdown of both isoforms. After finding that WHSC1L1 expression was required for typical proliferation rates of SUM44 cells, we performed genome-wide expression profiling of SUM44 WHSC1L1-short and total WHSC1L1 knockdown cell lines relative to a control SUM44 line transduced with shRNA against lacZ. Again we found that knockdown of the WHSC1L1-short alone had a greater affect than total WHSC1L1 knockdown, this time on the number of significantly differentially expressed genes; 1131 genes were found to be differentially expressed in the WHSC1L1-short knockdown cells relative to shLacZ control, while 238 genes were differentially expressed in the total WHSC1L1 knockdown SUM44 cells relative to shLacZ control. Interestingly, the ESR1 gene, which codes for the estrogen receptor alpha protein, was significantly downregulated by WHSC1L1-short knockdown. This was confirmed by immunoblotting with ERa antibody. While total WHSC1L1 knockdown also had a negative effect on ERa protein levels in SUM44, knockdown of WHSC1L1-short alone almost completely abrogated ERa in SUM44 as measured by western blot. We subsequently found that SUM44 cells were extremely sensitive to treatment with beta-estradiol, and that proliferation actually decreased upon as little as 100 picomolar beta-estradiol treatment, with dose-dependent decreases in proliferation as estrogen concentrations increased. We also found SUM44 cells to be relatively insensitive to Tamoxifen. Knockdown of WHSC1L1-short reduced proliferation of SUM44 cells in estrogen-free conditions, and treatment of SUM44 shWHSC1L1-short cells with increasing concentrations of beta-estradiol resulted in a marginal increase in proliferation up to 100pM beta-estradiol, then proliferation decreased with increasing beta-estradiol concentrations similar to results seen in SUM44 shLacZ control cells. After observing that WHSC1L1-short overexpression was required for expression of ERa in SUM44 cells, we asked whether knockdown of WHSC1L1-short affected genome-wide binding patterns of the estrogen receptor in SUM44 cells. Interestingly we found that ERa was binding to thousands of genomic loci in the absence of exogenous estrogen. Treatment with high doses (10nM) of beta-estradiol for 45 minutes resulted in an approximately even increase in ERa binding across sites already bound in the absence of estrogen, with some additional weak binding sites, but no significant changes in the pattern of ERa binding. No ERa binding sites were detected in SUM44 shWHSC1L1-short cells under estrogen-free conditions, and weak ERa binding was detected in SUM44 shWHSC1L1-short cells treated with 10nM beta-estradiol at loci where strong ERa binding was observed in control SUM44 cells, suggesting that WHSC1L1-short knockdown was reducing ERa expression levels, which made less ERa available to bind to chromatin in SUM44 shWHSC1L1-short cells. Our investigation of WHSC1L1 oncogenic activity in SUM44 cells resulted in the interesting observation that the short isoform of WHSC1L1 is required for expression of the estrogen receptor alpha in these cells, and that ERa is bound extensively to chromatin without activation of ERa by estrogen. SUM44 is a model for luminal B breast cancer, and is highly ER-positive, and expresses little to no progesterone receptor (PR). While the implications of ERa expression dependence on WHSC1L1-short overexpression in SUM44 cells are not yet clear, the extensive binding of ERa to estrogen response elements (ERE\u27s) in the absence of exogenous estrogen and the negative proliferative response of SUM44 to estrogen indicate that WHSC1L1 amplification and overexpression may alter the biology of the estrogen receptor in breast cancers harboring WHSC1L1 amplification and overexpression. Additionally, the differences seen in ERa binding to chromatin and the negative response of SUM44 cells to ERa agonists illustrate the importance of researching ER-positive breast cancer using additional cell line models rather than consistently using MCF7 cells to represent ER-positive disease. The dominant role of the catalytically-inactive short isoform of WHSC1L1 in regulating ERa expression and maintaining the proliferation rate of SUM44 cells suggests that catalytically inactive isoforms of chromatin modifying enzymes can be important regulators of gene expression. Interestingly, recent work has shown that WHSC1L1-short is likely not regulating target gene expression through histone methylation, but instead is acting as a co-factor for a different chromatin-binding complex, the BRD4-CHD8 complex, which has been shown to be recruited to superenhancer regions (marked by histone acetylation) by WHSC1L1-short, which results in activation of pTEFb through BRD4 and directly activates target gene transcription. It will be important to avoid assumptions about binding substrate identities of catalytically inactive isoforms of future chromatin modifiers of interest, as the catalytically inactive isoforms of these genes may also bind to chromatin substrates unrelated to the substrate of the catalytically active isoform

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Comprehensive genomic characterization of head and neck squamous cell carcinomas

The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCsclose9

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least 4 m. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the 6.5 m James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 yr, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit