| 1. |
- Birgersson, Madeleine, et al.
(författare)
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Antibody Validation for Estrogen Receptor Beta
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 1-23, s. 1-23
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies can cross-react with proteins other than their intended targets, and antibody-based applications can, if not properly validated, lead to flawed interpretations. When evaluating 13 anti-estrogen receptor beta (ERβ) antibodies in 2017, we concluded that only one of them was specific. Applying this antibody in immunohistochemistry of over 44 different normal human tissues and 20 types of cancers revealed ERβ expression in only a few selected tissues. This aligned with mRNA evidence but contradicted a large set of published literature. ERβ protein expression continues to be reported in tissues without clear support by mRNA expression. In this chapter, we describe how ERβ antibodies can be thoroughly validated and discuss selection of well-characterized positive and negative controls. The validation scheme presented is applicable for immunohistochemistry and Western blotting. The protocol includes evaluation of mRNA evidence, use of public databases, assessment of on- and off-target binding, and an optional step for corroboration with immunoprecipitation and mass spectrometry.
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| 2. |
- Dods, Robert, 1989, et al.
(författare)
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Ultrafast structural changes within a photosynthetic reaction centre.
- 2021
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 589:7841, s. 310-314
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.
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| 3. |
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| 4. |
- Archer, Amena, et al.
(författare)
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Expression Profiles of Estrogen-Regulated MicroRNAs in Cancer Cells.
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 313-343
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs play critical roles through their impact on posttranscriptional gene regulation. In cancer, they can act as oncogenes or tumor suppressors and can also function as biomarkers. Here, we describe a method for robust characterization of estrogen-regulated microRNA profiles. The activity of estrogen is mediated by two nuclear receptors, estrogen receptor alpha and estrogen receptor beta, and a transmembrane G-protein coupled estrogen receptor 1. This chapter details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, different microRNA profiling approaches, and subsequent confirmations.
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| 5. |
- Birgersson, Madeleine, et al.
(författare)
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ERβ in Granulosa Cell Tumors and Its Clinical Potential
- 2023
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Ingår i: Endocrinology. - : The Endocrine Society. - 0013-7227 .- 1945-7170. ; 164:6
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Forskningsöversikt (refereegranskat)abstract
- Granulosa cell tumors (GCTs) are rare ovarian tumors comprising an adult and a juvenile subtype. They have a generally good prognosis, but the survival rate drastically declines in patients with late-stage or recurring tumors. Due to the rarity of GCTs, the tumor type is largely understudied and lacks a specific treatment strategy. Estrogen receptor beta (ERβ/ESR2) has been found to be highly expressed in GCTs, which could be of therapeutic importance since it can be targeted with small molecules. However, its role in GCTs is not known. In this review, we summarize the current knowledge about the action of ERβ in the ovary and discuss its prospective role in GCTs.
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| 6. |
- Birgersson, Madeleine, et al.
(författare)
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Ovarian ERβ cistrome and transcriptome reveal chromatin interaction with LRH-1
- 2023
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Ingår i: BMC Biology. - : Springer Nature. - 1741-7007. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Estrogen receptor beta (ERβ, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized.Results: We here performed ERβ ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERβ knockout ovaries. By integrating the ERβ cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERβ loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1, Nr5a2) and showed that ERβ and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERβ and LRH-1 co-binding at the ERβ-repressed gene Greb1 but not at the ERβ-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERβ and LRH-1 can inhibit their respective transcriptional activity at classical response elements.Conclusions: By characterizing the genome-wide endogenous ERβ chromatin binding, gene regulations, and extensive crosstalk between ERβ and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.
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| 7. |
- Birgersson, Madeleine
(författare)
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Role and mechanism of estrogen receptor beta in the ovary and colon
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Estrogen regulates a variety of important physiological functions in both males and females, where the regulation of female reproduction and the development of sexual organs are typical examples. The effects of estrogen are predominantly exerted via signaling through the two nuclear receptors estrogen receptor α (ERα) and β (ERβ), or the membrane G protein-coupled estrogen receptor 1 (GPER1). While estrogen signaling is important for human health, dysregulation of signaling can have adverse effects and impact the development and progression of a wide range of diseases including reproductive disorders and cancer.ERβ has been shown to be highly important for ovarian function by regulating folliculogenesis and ovulation but has also been implied to protect against the development of colorectal cancer (CRC) by mediating the effects of estrogen. Despite the known role of ERβ, there is a lack of mechanistic understanding regarding how ERβ acts under both normal conditions and during disease. The overall aim of this thesis was to characterize the function and molecular mechanism of endogenous ERβ and to decipher its role in the normal ovary as well as its impact on colitis and CRC development. To further understand the role of estrogen signaling in the colon, we also aimed to identify sex differences during CRC development. In paper I we characterized the full cistrome of endogenous ovarian ERβ in the mouse and explored its transcriptional impact. We confirmed a direct role for ERβ in the regulation of essential ovarian functions and identified a novel crosstalk with the nuclear receptor LRH-1. In paper II we induced colitis-associated CRC (CAC) in intestinal epithelial-specific ERβ knockout mice and identified a protective effect by intestinal ERβ against tumor development in both male and female mice. We further characterized sex-dependent effects and proposed an underlying mechanism involving the regulation of TNFα/NFκB signaling. In paper III we expanded the investigation of sex-dependent changes during chemically-induced colitis in wildtype mice and identified a sex-specific response related to inflammatory response. We further found that male mice have an enhanced response to induced colitis.In paper IV the transcriptome of colitis-induced tumors and their immune cell infiltration was explored in wildtype and intestinal epithelial-specific ERβ knockout mice of both sexes. This showed that sex differences in the transcriptome appear to be dependent on the expression of ERβ. Also, the identified ERβ-dependent changes in the tumor transcriptome of female mice were specifically related to immune response. We corroborated an impact of ERβ on the infiltration of immune cells, especially a reduction of regulatory T cell and NK cell recruitment. In summary, this thesis provides new mechanistic understanding of the transcriptional role of ERβ in the normal ovary and in the colon microenvironment. This includes the discovery of crosstalk with LRH-1 in the ovary and NFκB in the colon. Our characterization provides a foundation to develop targeted therapies for improved fertility and chemoprevention in CRC. This thesis also highlights the importance of including both sexes in colitis and CRC research to advance our knowledge and improve treatment development.
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| 8. |
- Cheng, Yirui, et al.
(författare)
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Comparison of serum exosome isolation methods on co-precipitated free microRNAs.
- 2020
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Ingår i: PeerJ. - : PeerJ. - 2167-8359. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background: Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.Methods: In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods.Results: Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.
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| 9. |
- Cheng, Yirui, et al.
(författare)
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NDFIP1 limits cellular TAZ accumulation via exosomal sorting to inhibit NSCLC proliferation
- 2023
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Ingår i: Protein & Cell. - : Springer Nature. - 1674-800X .- 1674-8018. ; 14:2, s. 123-136
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Tidskriftsartikel (refereegranskat)abstract
- NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.
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| 10. |
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| 11. |
- Hakim Jaffer Ali, Mohammed, et al.
(författare)
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Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater
- 2021
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Ingår i: Science of the Total Environment. - : Elsevier BV. - 0048-9697 .- 1879-1026. ; 755
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Tidskriftsartikel (refereegranskat)abstract
- Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.
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| 12. |
- Hases, Linnea, et al.
(författare)
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Colitis Induces Sex-Specific Intestinal Transcriptomic Responses in Mice
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:18, s. 10408-
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Tidskriftsartikel (refereegranskat)abstract
- There are significant sex differences in colorectal cancer (CRC), including in incidence, onset, and molecular characteristics. Further, while inflammatory bowel disease (IBD) is a risk factor for CRC in both sexes, men with IBD have a 60% higher risk of developing CRC compared to women. In this study, we investigated sex differences during colitis-associated CRC (CAC) using a chemically induced CAC mouse model. The mice were treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) and followed for 9 and 15 weeks. We performed RNA-sequencing of colon samples from males (n = 15) and females (n = 15) to study different stages of inflammation and identify corresponding transcriptomic sex differences in non-tumor colon tissue. We found a significant transcriptome response to AOM/DSS treatment in both sexes, including in pathways related to inflammation and cell proliferation. Notably, we found a stronger response in males and that male-specific differentially expressed genes were involved in NF kappa B signaling and circadian rhythm. Further, an overrepresented proportion of male-specific gene regulations were predicted to be targets of Stat3, whereas for females, targets of the glucocorticoid receptor (Gr/Nr3c1) were overrepresented. At 15 weeks, the most apparent sex difference involved genes with functions in T cell proliferation, followed by the regulation of demethylases. The majority of sex differences were thus related to inflammation and the immune system. Our novel data, profiling the transcriptomic response to chemically induced colitis and CAC, indicate clear sex differences in CRC initiation and progression.
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| 13. |
- Hases, Linnea, et al.
(författare)
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ERβ and Inflammation
- 2022
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Ingår i: Advances in Experimental Medicine and Biology. - Cham : Springer Nature. ; 1390, s. 213-225
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Estrogen, through the regulation of cytokine production, can act both as pro-inflammatory and anti-inflammatory signals dependent on the tissue context. In breast cancer cells, ERα is known to modulate inflammatory signaling through interaction with NFκB. Whether ERβ has a role in inflammation is less explored. Low levels of ERβ have been corroborated in several immune-related organs and, for example, in colonic epithelial cells. Specifically, an impact of ERβ on colitis and colitis-associated colorectal cancer (CRC) is experimentally supported, using ERβ-selective agonists, full-body ERβ knockout mice and, most recently, intestinal epithelial-specific knockout mice. An intricate crosstalk between ERβ and TNFα/NFκB signaling in the colon is supported, and ERβ activation appears to reduce macrophage infiltration also during high fat diet (HFD)-induced colon inflammation. Finally, the gut microbiota plays a fundamental role in the pathogenesis of colitis and ERβ has been indicated to modulate the microbiota diversity during colitis and colitis-induced CRC. ERβ is thus proposed to protect against colitis, by modulating NFκB signaling, immune cell infiltration, and/or microbiota composition. Selective activation of ERβ may therefore constitute a suitable preventative approach for the treatment of for example colitis-associated CRC.
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| 14. |
- Hases, Linnea
(författare)
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Estrogen signaling in colon inflammation and colorectal cancer
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is the third most deadly form of cancer in the Western world. Although screening efforts have reduced the overall mortality, the incidence is increasing among young adults. The frequency of inflammatory bowel disease (IBD) and obesity are increasing in parallel, which suggest a common underlying environmental link between the conditions. This increase is thought to correlate to an increased intake of high fat diets, and obesity is a major risk factor for CRC. Chronic inflammation, which is a hallmark for CRC promotion, is a well-known underlying factor in both obesity and IBD. The gut microbiota is another hallmark, and an impaired relationship between the host and gut microbes can contribute to obesity, IBD and CRC. The risk-benefit balance of current CRC-preventative treatments is poor, and there is a need for safer and better preventatives in order to reduce the CRC mortality. Both obesity and IBD place men at a significant higher risk of CRC compared to women. This indicates a protective role for estrogen. The use of full Estrogen receptor (ER) β knockout mice has demonstrated ERβ protective effects against experimentally induced CRC. However, it is unknown through which cells these protective effects are mediated. There are only low mRNA levels of ERβ in the colon, unclear if adequate for a functional role, and ERβ may also be expressed in intestinal immune cells. Understanding the CRC-preventative effects of intestinal epithelial ERβ in both sexes is important and may provide the background for a novel CRC chemopreventive approach.The overall aim of the thesis is the functional characterization of intestinal epithelial ERβ during colon inflammation and colitis-induced CRC and identification of potential sex differences, which can ultimately provide novel opportunities for chemopreventive exploitation (Figure 1). In paper I we utilized intestinal epithelial ERβ knockout mice (ERβKOVil) of both sexes and induced colitis and colitis associated CRC (CA-CRC). We found that intestinal epithelial ERβ is protective against colitis and CA-CRC in both sexes, but in a sex-dependent manner. The underlying mechanism includes an intricate crosstalk with TNFα-induced NFκB signaling.In paper II we identify that both sex and intestinal epithelial ERβ impact the microbiota composition. This may contribute to the exacerbated colitis and colitis-induced tumor formation observed in ERβKOVil mice.In paper III we induced colon inflammation by feeding the mice a high-fat diet (HFD, 60%) for 13 weeks and explored treatment with estrogen receptor-selective ligands. We identified that estrogen signaling, via ERβ, modulated the HFD-induced changes in the colon microenvironment. This included sex-dependent effects on epithelial cell proliferation, macrophage infiltration, and regulation of core circadian clock gene expression.In paper IV we utilized paired-normal and CRC clinical samples and identified sex differences in the transcriptome of both normal colon and CRC. By applying data-driven feature selection and machine learning on sex-separated TCGA data, we proposed sex-specific diagnostic biomarkers and prognostic biomarkers using survival analysis. In summary, this thesis characterizes intestinal epithelial ERβ as a novel chemopreventative target for CA-CRC in both sexes, and identifies related biological pathways, including crosstalk with nuclear factor κB (NFκB) signaling and modulation of circadian clock genes. ERβ activity in intestinal epithelial cells is manifested by altered microbiota composition, cell proliferation and immune cell infiltration. The identification of several significant sex differences provides evidence for the need to take sex into account in colitis and CRC research to improve health interventions.
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| 15. |
- Hases, Linnea, et al.
(författare)
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High-fat diet and estrogen impacts the colon and its transcriptome in a sex-dependent manner
- 2020
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- There is a strong association between obesity and colorectal cancer (CRC), especially in men, whereas estrogen protects against both the metabolic syndrome and CRC. Colon is the first organ to respond to high-fat diet (HFD), and estrogen receptor beta (ERβ) can attenuate CRC development. How estrogen impacts the colon under HFD and related sex differences has, however, not been investigated. To dissect this, mice were fed control diet or HFD for 13 weeks and administered receptor-selective estrogenic ligands for the last three weeks. We recorded impact on metabolism, colon crypt proliferation, macrophage infiltration, and the colon transcriptome. We found clear sex differences in the colon transcriptome and in the impact by HFD and estrogens, including on clock genes. ERα-selective activation reduced body weight and generated systemic effects, whereas ERβ-selective activation had local effects in the colon, attenuating HFD-induced macrophage infiltration and epithelial cell proliferation. We here demonstrate how HFD and estrogens modulate the colon microenvironment in a sex- and ER-specific manner.
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| 16. |
- Hases, Linnea, et al.
(författare)
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High-fat diet and estrogen modulate the gut microbiota in a sex-dependent manner in mice
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- A high-fat diet can lead to gut microbiota dysbiosis, chronic intestinal inflammation, and metabolic syndrome. Notably, resulting phenotypes, such as glucose and insulin levels, colonic crypt cell proliferation, and macrophage infiltration, exhibit sex differences, and females are less affected. This is, in part, attributed to sex hormones. To investigate if there are sex differences in the microbiota and if estrogenic ligands can attenuate high-fat diet-induced dysbiosis, we used whole-genome shotgun sequencing to characterize the impact of diet, sex, and estrogenic ligands on the microbial composition of the cecal content of mice. We here report clear host sex differences along with remarkably sex-dependent responses to high-fat diet. Females, specifically, exhibited increased abundance of Blautia hansenii, and its levels correlated negatively with insulin levels in both sexes. Estrogen treatment had a modest impact on the microbiota diversity but altered a few important species in males. This included Collinsella aerofaciens F, which we show correlated with colonic macrophage infiltration. In conclusion, male and female mice exhibit clear differences in their cecal microbial composition and in how diet and estrogens impact the composition. Further, specific microbial strains are significantly correlated with metabolic parameters.
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| 17. |
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| 18. |
- Hases, Linnea, et al.
(författare)
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Intestinal estrogen receptor beta suppresses colon inflammation andtumorigenesis in both sexes
- 2020
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Ingår i: Cancer Letters. - : Elsevier BV. - 0304-3835 .- 1872-7980. ; 492, s. 54-62
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Tidskriftsartikel (refereegranskat)abstract
- Estrogen hormones protect against colorectal cancer (CRC) and a preventative role of estrogen receptor beta (ERβ) on CRC has been supported using full knockout animals. However, it is unclear through which cells or organ ERβ mediates this effect. To investigate the functional role of intestinal ERβ during colitis-associated CRC we used intestine-specific ERβ knockout mice treated with azoxymethane and dextran sodium sulfate, followed by ex vivo organoid culture to corroborate intrinsic effects. We explored genome-wide impact on TNFα signaling using human CRC cell lines and chromatin immunoprecipitation assay to mechanistically characterize the regulation of ERβ. Increased tumor formation in males and tumor size in females was noted upon intestine-specific ERβ knockout, accompanied by enhanced local expression of TNFα, deregulation of key NFκB targets, and increased colon ulceration. Unexpectedly, we noted especially strong effects in males. We corroborated that intestinal ERβ protects against TNFα-induced damage intrinsically, and characterized an underlying genome-wide signaling mechanism in CRC cell lines whereby ERβ binds to cis-regulatory chromatin areas of key NFκB regulators. Our results support a protective role of intestinal ERβ against colitis-associated CRC, proposing new therapeutic strategies.
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| 19. |
- Hases, Linnea, et al.
(författare)
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The Importance of Sex in the Discovery of Colorectal Cancer Prognostic Biomarkers
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:3
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Tidskriftsartikel (refereegranskat)abstract
- Colorectal cancer (CRC) is the third leading cause of cancer deaths. Advances within bioinformatics, such as machine learning, can improve biomarker discovery and ultimately improve CRC survival rates. There are clear sex differences in CRC characteristics, but the impact of sex has not been considered with regards to CRC biomarkers. Our aim here was to investigate sex differences in the transcriptome of a normal colon and CRC, and between paired normal and tumor tissue. Next, we attempted to identify CRC diagnostic and prognostic biomarkers and investigate if they are sex-specific. We collected paired normal and tumor tissue, performed RNA-seq, and applied feature selection in combination with machine learning to identify the top CRC diagnostic biomarkers. We used The Cancer Genome Atlas (TCGA) data to identify sex-specific CRC diagnostic biomarkers and performed an overall survival analysis to identify sex-specific prognostic biomarkers. We found transcriptomic sex differences in both the normal colon tissue and in CRC. Forty-four of the top-ranked biomarkers were sex-specific and 20 biomarkers showed a sex-specific prognostic value. Our data show the importance of sex in the discovery of CRC biomarkers. We propose 20 sex-specific CRC prognostic biomarkers, including ESM1, GUCA2A, and VWA2 for males and CLDN1 and FUT1 for females.
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| 20. |
- Huang, Dan, et al.
(författare)
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Estrogen Receptor beta (ESR2) Transcriptome and Chromatin Binding in a Mantle Cell Lymphoma Tumor Model Reveal the Tumor-Suppressing Mechanisms of Estrogens
- 2022
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 14:13, s. 3098-
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Tidskriftsartikel (refereegranskat)abstract
- Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with one of the highest male-tofemale incidence ratios. The reason for this is not clear, but epidemiological as well as experimental data have suggested a role for estrogens, particularly acting through estrogen receptor beta (ESR2). To study the ESR2 effects on MCL progression, MCL cells sensitive and resistant to the Bruton tyrosine kinase inhibitor ibrutinib were grafted to mice and treated with the ESR2-selective agonist diarylpropionitrile (DPN). The results showed that the DPN treatment of mice grafted with both ibrutinib-sensitive and -resistant MCL tumors resulted in impaired tumor progression. To identify the signaling pathways involved in the impaired tumor progression following ESR2 agonist treatment, the transcriptome and ESR2 binding to target genes were investigated by genome-wide chromatin immunoprecipitation in Granta-519 MCL tumors. DPN-regulated genes were enriched in several biological processes that included cell-cell adhesion, endothelial-mesenchymal transition, nuclear factor-kappaB signaling, vasculogenesis, lymphocyte proliferation, and apoptosis. In addition, downregulation of individual genes, such as SOX11 and MALAT1, that play a role in MCL progression was also observed. Furthermore, the data suggested an interplay between the lymphoma cells and the tumor microenvironment in response to the ESR2 agonist. In conclusion, the results clarify the mechanisms by which estrogens, via ESR2, impair MCL tumor progression and provide a possible explanation for the sex-dependent difference in incidence. Furthermore, targeting ESR2 with a selective agonist may be an additional option when considering the treatment of both ibrutinib-sensitive and -resistant MCL tumors.
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| 21. |
- Indukuri, Rajitha, et al.
(författare)
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An Optimized ChIP-Seq Protocol to Determine Chromatin Binding of Estrogen Receptor Beta.
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 203-221
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Tidskriftsartikel (refereegranskat)abstract
- Estrogen regulates transcription through two nuclear receptors, ERα and ERβ, in a tissue and cellular-dependent manner. Both the receptors bind estrogen and activate transcription through direct or indirect interactions with DNA. Revealing their interactions with the chromatin is key to understanding their transcriptional activities and their biological functions. Chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful technique to map protein-DNA interactions at precise genomic locations. The genome-wide binding of ERα has been extensively studied. Similar studies of ERβ, however, have been more difficult, in part due to a lack of endogenous expression in cell lines and lack of specific antibodies. In this chapter, we provide an optimized stepwise ChIP protocol for a well-validated ERβ antibody, which is applicable for ChIP-Seq analysis of cell lines with exogenous expression of ERβ.
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| 22. |
- Indukuri, Rajitha, et al.
(författare)
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Estrogen Receptor Beta Influences the Inflammatory p65 Cistrome in Colon Cancer Cells
- 2021
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Ingår i: Frontiers in Endocrinology. - : FRONTIERS. - 1664-2392. ; 12, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- Inflammation is a primary component of both initiation and promotion of colorectal cancer (CRC). Cytokines secreted by macrophages, including tumor necrosis factor alpha (TNFα), activates the pro-survival transcription factor complex NFκB. The precise mechanism of NFκB in CRC is not well studied, but we recently reported the genome-wide transcriptional impact of TNFα in two CRC cell lines. Further, estrogen signaling influences inflammation in a complex manner and suppresses CRC development. CRC protective effects of estrogen have been shown to be mediated by estrogen receptor beta (ERβ, ESR2), which also impacts inflammatory signaling of the colon. However, whether ERβ impacts the chromatin interaction (cistrome) of the main NFκB subunit p65 (RELA) is not known. We used p65 chromatin immunoprecipitation followed by sequencing (ChIP-Seq) in two different CRC cell lines, HT29 and SW480, with and without expression of ERβ. We here present the p65 colon cistrome of these two CRC cell lines. We identify that RELA and AP1 motifs are predominant in both cell lines, and additionally describe both common and cell line-specific p65 binding sites and correlate these to transcriptional changes related to inflammation, migration, apoptosis and circadian rhythm. Further, we determine that ERβ opposes a major fraction of p65 chromatin binding in HT29 cells, but enhances p65 binding in SW480 cells, thereby impacting the p65 cistrome differently in the two cell lines. However, the biological functions of the regulated genes appear to have similar roles in both cell lines. To our knowledge, this is the first time the p65 CRC cistrome is compared between different cell lines and the first time an influence by ERβ on the p65 cistrome is investigated. Our work provides a mechanistic foundation for a better understanding of how estrogen influences inflammatory signaling through NFκB in CRC cells.
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| 23. |
- Indukuri, Rajitha
(författare)
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Estrogen receptor beta transcriptional regulation: A potential mechanism for colon cancer prevention
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is the third leading cause of death from cancer in both men andwomen in the Western world. Improved screening efforts, surveillance, and treatment havereduced CRC mortality in older patients. However, the incidence is increasing in young adults,even in the absence of CRC family history. This may indicate an influence of increasingobesity, changed dietary patterns, and lifestyle factors. The progression of CRC is a multistepprocedure that takes 10-15 years, thus offering a time to implement preventative measures andearly detection. There is a critical need to develop more effective preventive therapies due tothe risks posed by current prevention therapies. The best CRC prophylactic agent should beboth safe and suitable to use for a long time (1).In preclinical studies, estrogen has been shown to protect from CRC, and substantial evidencesuggests it is through estrogen receptor beta (ERβ). Natural ERβ selective agonists have beentested in phase II clinical trials to treat menopause symptoms and proven to be safe and welltolerated with no side effects (2, 3). Thus, selective activation of ERβ with selective agonists,which do not activate estrogen receptor alpha (ERα), is a potential clinical approach inpreventing adenomatous polyps progression into CRC. However, the mechanism of thesebeneficial ERβ effects is not well understood, and there is a significant knowledge gap in thisarea.The overall aim of this thesis was to identify the mechanistic background of the intestinal ERβmediated antitumorigenic effects in the colon and further to explore ERβ as a preventativeapproach in CRC. One specific aim was to determine whether ERβ present specifically in colonepithelium is responsible for protecting from CRC, which is addressed in Paper I. Tounderstand the impact of ERβ in protecting from colitis-associated CRC (CA-CRC), we haveinduced colitis in intestinal-specific ERβ knockout mice of both sexes. The loss of intestinalERβ aggravated CA-CRC in a sex-dependent manner. The incidence of tumors increased inmales, while in females, the size of the tumors was enhanced. We identified that ERβ attenuatestumor necrosis factor alpha (TNFα) induced epithelial cell damage and modulates theregulation of key nuclear factor-κB (NFκB) members. As a direct consequence, ERβ was foundto reduce inflammation and to control intestinal crypt cell proliferation.Another aim was to explore transcriptional regulation by ERβ through mapping of chromatinbinding sites and interaction with NFκB, which is studied in Paper II and IV. Commonly usedERβ antibodies have been shown to be unspecific towards ERβ; this study used a validatedERβ antibody to map genome-wide ERβ binding sites in colon cancer cells. We observed thatthe presence of ERβ also modulated the regulatory chromatin mark H3K27AC in potentialenhancers of transcriptional regulation, Wnt signaling, and cell proliferation. Further, motifanalysis indicated a novel ERβ colon-specific cross-talk with TCF, and KLF motifs supporteda interaction between β-catenin/TCF and ERβ. We found that ERβ binds and regulates severalimportant tumor suppressors and oncogenes in CRC cells, such as CST5 and LRP6, consistentwith its proposed antitumorigenic activity. We also revealed the p65 cistrome in CRC cell lines and showed that ERβ alters the p65 chromatin binding in a cell-type-dependent manner. Wefound that ERβ chromatin binding sites were enriched among circadian clock genes and alsothat ERβ modulates p65 binding to core clock genes in CRC cells, indicating potential crosstalk between ERβ and circadian clock gene regulation.The final aim was to investigate the impact of ERβ, and estrogen signaling in high-fat diet(HFD) induced inflammation in colon, explored in paper III. We fed mice with an HFD for 13weeks and treated them with estrogenic ligands for the last three weeks prior to sacrifice. Thecolon transcriptome showed predominant sex differences, and selective activation of ERβreduced macrophage infiltration and epithelial cell proliferation induced by HFD. Wedemonstrated that ERβ opposes HFD-induced dysregulation of core circadian clock genes invivo, further strengthening the role of ERβ in circadian rhythm.Taken together, these results highlight the chemopreventive potential of ERβ in CRC in bothsexes. The identified cross-talk with TNFα/NFκB pathway, Wnt signaling, regulating genesinvolved circadian clock, and tumorigenesis reflected ERβ protection/antitumor activityagainst CRC progression and development (as illustrated in Figure 1).
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- Indukuri, Rajitha, et al.
(författare)
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Genome-wide estrogen receptor β chromatin binding in humancolon cancer cells reveals its tumor suppressor activity
- 2021
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215.
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Tidskriftsartikel (refereegranskat)abstract
- Colorectal cancer (CRC) is the third leading cause of cancer death in the western world. In women, menopausal hormone therapy has been shown to reduce CRC incidence by 20%. Studies demonstrate that estrogen activating estrogen receptor beta (ERβ) protects against CRC. ERβ is a nuclear receptor that regulates gene expression through interactions with the chromatin. This molecular mechanism is, however, not well characterized in colon. Here, we present for the first time, the cistrome of ERβ in different colon cancer cell lines. We use cell lines engineered to express ERβ, optimize and validate an ERβ antibody for chromatin-immunoprecipitation (ChIP), and perform ChIP-Seq. We identify key binding motifs, including ERE, AP-1, and TCF sites, and we determine enrichment of binding to cis-regulatory chromatin sites of genes involved in tumor development, cell migration, cell adhesion, apoptosis, and Wnt signaling pathways. We compare the corresponding cistromes of colon and breast cancer and find that they are conserved for about a third of genes, including GREB1, but that ERβ tethering to TCF and KLF family motifs is characteristic for colon. We exemplify upregulation of putative CRC tumor suppressor gene CST5 where ERβ in colon cells binds to cis-regulatory regions nearby (−351 bp) the transcriptional start site. Our work provides a foundation for understanding the mechanism of action of ERβ in CRC prevention.
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