A combined proteomics and Mendelian randomization approach to investigate the effects of aspirin-targeted proteins on colorectal cancer
TL;DR: MCM6 and RRM2 are involved in DNA repair whereby reduced expression may lead to increased DNA aberrations and ultimately cancer cell death, whereas ARFIP2 is involved in actin cytoskeletal regulation indicating a possible role in aspirin’s reduction of metastasis.
Abstract: Background: Evidence for aspirin9s chemopreventative properties on colorectal cancer (CRC) is substantial, but its mechanism of action is not well-understood. We combined a proteomic approach with Mendelian randomization (MR) to identify possible new aspirin targets that decrease CRC risk. Methods: Human colorectal adenoma cells (RG/C2) were treated with aspirin (24 hours) and a stable isotope labelling with amino acids in cell culture (SILAC) based proteomics approach identified altered protein expression. Protein quantitative trait loci (pQTLs) from INTERVAL (N=3,301) and expression QTLs (eQTLs) from the eQTLGen Consortium (N=31,684) were used as genetic proxies for protein and mRNA expression levels. Two-sample MR of mRNA/protein expression on CRC risk was performed using eQTL/pQTL data combined with CRC genetic summary data from the Colon Cancer Family Registry (CCFR), Colorectal Transdisciplinary (CORECT), Genetics and Epidemiology of Colorectal Cancer (GECCO) consortia and UK Biobank (55,168 cases and 65,160 controls). Results: Altered expression was detected for 125/5886 proteins. Of these, aspirin decreased MCM6, RRM2 and ARFIP2 expression and MR analysis showed that a standard deviation increase in mRNA/protein expression was associated with increased CRC risk (OR:1.08, 95% CI:1.03-1.13, OR:3.33, 95% CI:2.46-4.50 and OR:1.15, 95% CI:1.02-1.29, respectively). Conclusion: MCM6 and RRM2 are involved in DNA repair whereby reduced expression may lead to increased DNA aberrations and ultimately cancer cell death, whereas ARFIP2 is involved in actin cytoskeletal regulation indicating a possible role in aspirin9s reduction of metastasis. Impact: Our approach has shown how laboratory experiments and population-based approaches can combine to identify aspirin-targeted proteins possibly affecting CRC risk.
Summary (3 min read)
- Several reports indicate that females have a stronger immune response, partly due to differences in hormonal profile [1, 2].
- Recently, TTV has received attention as a possible endogenous biomarker for immune function, with immunocompetent individuals carrying lower levels of TTV in serum than immunocompromised, indicating a suppressing role of the immune system on the viral load .
- The high turnover-rate of virions indicates that changes in immune status can be followed in a short time frame.
- The differences in female and male immunity towards pathogens have implications for treatment and prevention of infectious diseases and may ultimately lead to a different approach depending on the sex of the patient.
- 27 healthy individuals were included according to a protocol approved by the Central Ethical Review Board (Swedish Research Council, Stockholm, Dnr: €O 24–2009) and consisted of 17 premenopausal women, 6 men and 4 postmenopausal women (Table 1).
- The subjects were included and sampled during 6 months (between March and September, 2010).
- Informed consent was obtained from the participants.
- Premenopausal women aged 20–40 years with regular menstrual cycles, without hormonal contraceptives or other hormonal, anti-inflammatory (including ASA, systemic cortisone and NSAIDs) or any morphine treatment since>3 months, and parturition no later than 12 months before inclusion, also known as The inclusion criteria were.
- Men (aged 20–70) and postmenopausal women (no menstrual bleeding since >12 months) without the above stated treatment during the last 3 months.
2.2. Blood sampling and hormonal analyses
- From all individuals blood was drawn at four timepoints, and for the pre-MP women Ovustick® was used to identify the LH-peak.
- Simultaneously, at one or more timepoints PBMC was also sampled.
- The buffycoat was transferred to new tubes and slowly frozen in 20% dimethylsulphoxide (DMSO)-albumin, using isopropanol-loaded Mr. Frosty® freezing-container overnight, before long-term storage at -80 C.
- Analyses were made of WBC, differential count (including B-monocytes, Blymphocytes, B-neutrophils, B-eosinophils, B-basophils), S-TSH (thyroid stimulating hormone), S-T4, S-SHBG (sex hormone binding globulin), Sestradiol, S-testosterone, S-progesterone, S-FSH, S-LH and S-prolactin.
- The participants were assessed for hypo- or hyperthyroidism, and preMP women also whether they had a normal ovulation.
2.3. TTV DNA isolation and analysis
- This was performed according to QIAamp® DNA Mini and Blood Mini Handbook to increase DNA yield.
- This contained pre-prepared solutions of 5, 50, 500 and 5000 copies plasmid TTV DNA per μL, as well as a sensitivity control containing 1 copy/μL.
- The sample wells were run in triplicates using 10 μL of concentrated DNA solution.
- According to the standard curve obtained, this corresponded to CT of 37.09, 42.09 and 39.14 respectively on the included three TTV qPCR plates.
- A sample was considered positive if 2 of 3 triplicate samples were above the detection limit (i.e. below the CT-threshold mentioned above).
2.4. Statistical analyses
- The average hormone levels of TSH, estradiol, LH and testosterone were calculated for each of the 17 pre-MP women.
- As a logistic model with logit-link, the following was used: TTV ~ log (Mean_TSH) þ log (Mean_estradiol) þ log (Mean_LH) þ log (Mean_testosterone).
- The explanatory variables are treated as covariates.
3.2. TTV prevalence and TTV levels
- The detected levels of TTVwere highest among the TTV positive pre-MPwomen and lower in the post-MP women and in the men, both in terms of detected TT viral copies/mL and when adjusting for total amount of DNA in the sample (Table 2).
- The differences in TTV prevalence between pre-MP and postMP women as well as between pre-MP women and men were not statistically significant (Fisher's exact test, p > 0.999 and p ¼ 0.2786) .
- The raw data suggested higher prevalence in men than in preMP women, but significance testing could not rule out a chance yroid status, sex hormone levels.
- For pre-MP women, day of the menstrual cycle, rone and LH, is indicated.
3.3. Hormonal status in TTV-positive pre-MP women
- To determine whether sex hormones influence the risk of being TTVpositive (TTVþ) the authors compared the average sex hormone levels in TTVþ (n ¼ 3) and TTV (n¼ 14) individuals using a binomial regression including S-estradiol, S-testosterone, S-LH and S-TSH.
- When comparing average TSH from TTVþ and TTV individuals in a binominal regression, there was no significant difference (p-value¼ 0.337, Table 3).
- Samples from themid-luteal phase revealed (using Welch's t-test) significantly lower progesterone levels (p ¼ 0.002) in TTVþ compared to TTV pre-MP women .
- Dispersion parameter for binominal family eviance: 5.5798 on 13 Df. Akaike information criterion (AIC): 15.58.
3.4. Hormonal status in men and post-MP women
- All three TTV men, and two out of three TTV post-MP women hade traces of TTV, but below cut-off (Table 4B).
- All men and post-MP women (TTVþ and TTV ) had normal levels of TSH.
- Due to the low number of participants, it was not possible to use regression models for analyzing hormone levels in relation to TTV status in men and post-MP women.
- TTV status, age and range of hormone levels are shown in Table 5.
- The detection of TTV in 17.6% pre-MP females, 25.0% post-MP females, and 50.0%males suggests that TTV presence in the PBMC fraction of peripheral blood may be associated with sex.
- TTVþ samples from pre-MP women in their cohort were mostly found during the first half of the menstrual cycle, and pre-MP women who were positive for TTV had hormonal aberrances being either anovulatory, hypothyroid or both.
- Interactions of sex hormones with the immune system are established on multiple levels (reviewed in e.g. [10, 15, 16]).
- In a study by Maggi et al. (2001), TTV-levels were considerably lower in PBMC than in plasma from the same individuals .
- Only 2.4% of the plasma samples in the study from Fern andez-Ruíz et al. , were below lower limit of detection, whereas in their study on PBMC, the majority of individuals (74.1%) were below cut-off.
Did you find this useful? Give us your feedback
"A combined proteomics and Mendelian..." refers background in this paper
...Colorectal cancer (CRC) is the fourth most common cancer worldwide (1)....
"A combined proteomics and Mendelian..." refers methods in this paper
...As found by the Genotype-Tissue Expression 658 (GTEx) study, cis eQTLs are either shared across tissues or are specific to a small number of tissues 659 (54)....
"A combined proteomics and Mendelian..." refers methods in this paper
...due to horizontal pleiotropy 507 (where a genetic variant affects the outcome through an alternative exposure/pathway of interest) 508 (17), then the estimator from the IVW method becomes biased (38)....
...In MR, genetic variants 403 (most commonly single nucleotide polymorphisms (SNPs)) are used to proxy an exposure of interest 404 (17)....
"A combined proteomics and Mendelian..." refers background or methods in this paper
...As a sensitivity analysis, 509 alternative MR methods were used when more than 2 SNPs were available as instruments for 510 mRNA/protein expression (MR Egger, simple mode, weighted mode, and weighted median) 511 (36,39,40)....
...The weighted median approach is useful as it allows a consistent estimate even if 50% of the 514 SNPs proxying protein/mRNA expression are invalid instruments (40) and the mode estimate also 515 provides a consistent causal effect estimate even if the majority of the instruments are invalid, as 516 the estimate depends on the largest number of similar instruments (39)....
Related Papers (5)
Frequently Asked Questions (2)
Q1. What have the authors contributed in "Blood hormones and torque teno virus in peripheral blood mononuclear cells" ?
In this study the authors investigated the presence and levels of TTV in peripheral blood mononuclear cells ( PBMCs ) in premenopausal ( pre-MP ) women, post-menopausal ( post-MP ) women, and men, and determined their serum sex hormone levels. Of the examined subjects ( n 1⁄4 27 ), the authors found presence of TTV in PMBC from 17. Although their study was performed on a limited number of subjects, the data suggests that TTV in PBMC is associated with an anovulatory menstrual cycle with low progesterone levels, and possibly with male sex.
Q2. What have the authors stated for future works in "Blood hormones and torque teno virus in peripheral blood mononuclear cells" ?
Preferably, future studies including sex and hormonal status ( e. g. pre- or post-menopause, contraceptives, hormonal replacement therapies and ovulation, as well as signs of hypothyroidism ), should be performed to obtain more information on the impact of the menstrual cycle on TTV load and immune response.