Night-shift work duration and risk of colorectal cancer according to IRS1 and IRS2 expression

Abstract

Objective:

We hypothesized that the risk of CRC in night-shift workers might be different according to insulin receptor substrates status.

Methods:

Among 77,470 eligible women having night work assessed in the Nurses’ Health Study, we documented a total of 1,397 CRC cases, of which 304 or 308 had available data on IRS1 and IRS2, respectively. We used duplication method Cox proportional hazards regression analysis for competing risks to calculate HRs and 95% CIs for each CRC subtype. We measured tumor IRS1 or IRS2 expression by immunohistochemistry.

Results:

Compared with women who never worked night-shifts, those working ≥ 15 years night-shifts had a marginal trend of increased overall risk of CRC (P_trend = 0.06, multivariable HR = 1.20, 95% CI, 0.99 to 1.45). Longer duration of night-shift work was associated with a higher risk of IRS2-positive tumors (multivariable HR = 2.69, 95% CI 1.48 to 4.89, P_trend = 0.001, ≥ 15 years night-shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90, 95% CI 0.54 to 1.51, P_trend = 0.72, P_{heterogeneity} for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI 0.94 to 3.48, P_trend = 0.06) and 1.13 for IRS1-negative tumors (95% CI 0.71 to 1.80, P_trend = 0.56, P_{heterogeneity} for IRS1 = 0.02).

Conclusion:

Our molecular pathological epidemiology data suggest a potential role of IRS in mediating carcinogenesis induced by night-shift work.

Impact:

Although these findings need validation, rotating night-shift might increase CRC risk in women with abnormal insulin receptor pathway.

INTRODUCTION

Shift work is considered as a “probable” (class 2A) carcinogen to human by the International Agency for Research on Cancer.(1,2) Accumulating evidence suggests that shift work involving circadian rhythm disruption is associated with increased risk of some types of cancers such as breast and colorectal cancer (CRC).(3,4) Schernhammer et al previously reported an increased CRC risk with longer duration of night-shift work in the Nurses’ Health study (NHS) in 2003.(4) Subsequent studies (3,5,6) including a recent meta-analysis (7) reported similar findings. However, other studies (8,9) including a most recent one from Papantoniou et al failed to replicate these findings.(9) It is conceivable that dealing with inherently heterogeneous CRC as a single entity might have diluted any risk association with shift work that might exist for a specific molecular subtype of CRC. Further exploring the underlying biological mechanisms would be helpful to better understand these inconsistent associations. Given that CRC is a highly heterogeneous disease, night-shift work might have different effects on the development of different subgroups of CRC defined by tumor molecular characteristics. Thus, molecular pathological epidemiology (MPE) approach that integrates molecular pathology into epidemiological research (10) can link a certain exposure (such as shift work) to specific pathological signatures, thereby better elucidating the possible pathogenic effect of night-shift work on CRC development.

Recent experimental studies suggested that circadian rhythm disruption may be associated with β-cell dysfunction, glucose intolerance, and improper insulin secretion.(11,12) Similarly, population studies showed that night shift worker tended to have lower insulin sensitivity, hyperinsulinemia, insulin resistance, and develop metabolic syndrome, accordingly.(13–17) Insulin resistance is an adaptive process in insulin-sensitive tissues characterized by reduced insulin receptor substrate 1 (IRS1), and increased IRS1 serine phosphorylation and attenuated downstream signaling. However, there is some evidence demonstrating that the presence of high insulin levels may not necessarily cause insulin resistance, but instead was associated with IRS1 or IRS2 expression and / or tyrosine phosphorylation, which could activate downstream the PI3K / MTOR pathway and subsequently promote mitogenesis and cell proliferation, as shown in colon cancer cells and mouse skeletal muscle cells.(18,19) In addition, human evidence reported positive association between high levels of insulin and risk of colon cancer.(20,21) Since IRS1 and IRS2 are two primary mediators of insulin-dependent mitogenesis and regulation of glucose metabolism in most cell types,(22) and abundantly expressed in CRC,(23) it appears plausible that IRS1 and IRS2 play a key role in colorectal carcinogenesis as part of the chronic metabolic disorder observed in night-shift workers.(24)

In light of this evidence, we hypothesized that longer duration of night-shift work might be associated with an increased risk of colorectal cancer overexpressing IRSs. To test our hypothesis, we prospectively investigated the association of duration of night-shift work with CRC risk according to tumor IRS1 or IRS2 expression in the Nurses’ Health Study (NHS).

In this cohort, we have previously found that women who worked rotating night-shifts for at least 15 years were at an increased risk of CRC.(4) Integrating host factors (such as night-shift work) and tumor molecular features (such as IRSs expression) may enhance our understanding of the mechanisms through which night-shift work may act on colorectal carcinogenesis.

METHODS

Study population and assessment of night-shift work duration

Participants were identified from the Nurses’ Health Study (NHS). Details for the study design and the population have been reported elsewhere.(25–27) A total of 121,700 female registered nurses aged 30 to 55 years were enrolled at baseline in 1976 in the U.S. A biennial questionnaire has been sent to all the participants since 1976 to collect updated information regarding demographics, lifestyle factors, and medical history. Returning the questionnaires was considered to imply informed consent. All procedures of the study were in accordance with the Declaration of Helsinki. The study protocol was approved by the institutional review boards at the Brigham and Women’s Hospital, Boston, Massachusetts.

As described previously,(4,28) NHS participants were asked “how many years have you worked rotating night-shifts including at least 3 nights per month, in addition to days or evenings in that month” in 1988. Information on lifetime years of rotating night-shift work was collected in 8 pre-specified categories, which are never, 1 - 2, 3 - 5, 6 - 9, 10 - 14, 15 - 19, 20 - 29, and ≥ 30 years. We excluded women with a history of any cancer other than non-melanoma skin cancer, polyposis syndrome, ulcerative colitis, or Crohn’s disease in or before 1988, or who did not report their night-shift work duration. A total of 77,470 women were included in this analysis. (Figure 1)

Figure 1.

Flow chart of the study population in the Nurses’ Health Study.

Assessment of covariates

We collected information on potential CRC risk factors including height, body weight, physical activity (METS-hours/week), cigarette smoking, history of sigmoidoscopy or colonoscopy screening, family history of colorectal cancer, history of type 2 diabetes, aspirin use, and menopausal status and use of menopausal hormones at baseline and updated in biennial follow-up questionnaires. Body mass index (BMI) was calculated based on reported height and weight. In addition, we collected information on dietary factors including consumption of alcohol, vitamin D, folate, calcium, red meat, processed meat using a validated food frequency questionnaire, with updates almost every 4 years.(29,30) Furthermore, in 1986, 2000, 2002 and 2008, we asked how many hours a woman slept, on average, in a 24-hour period (5 hours or less, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or 11 hours or more). Self-reported sleep duration correlated well with sleep duration assessed by sleep diaries in this cohort (Spearman r = 0.79; P < 0.0001).(31)

Ascertainment of incident colorectal cancer cases

Participants or next-of-kin were asked for written permission to obtain medical records and pathological reports if they reported cancer on biennial questionnaires. Study researchers blinded to exposure status further reviewed medical and pathological records to confirm all possible CRC cancer cases, and extracted the information on anatomic location, stage and histological type of the cancer. CRC cases were defined as primary tumors with International Classification of Diseases-9 (ICD-9) codes 153 and 154 and with the histological subtypes, adenocacinoma, signet-ring cell cancer, adenosquamous cancer, as well as undifferentiated cancer (excluding carcinoid, squamous cell cancer, and non-epithelial malignancies, such as sarcoma and lymphoma). We identified unreported fatal CRC and death from state vital statistics records and the National Death Index.

Immunohistochemistry for IRS1 and IRS2 expression

We collected formalin-fixed paraffin-embedded (FFPE) archival tissue specimens of colorectal carcinoma resections from hospitals and laboratories, and constructed tissue microarrays (TMA) from CRC blocks as previously described.(32) Methods for tumor IRS1 and IRS2 immunohistochemistry have been previously described.(33) TMA sections were deparaffinized, rehydrated, and heated in a pressure cooker for 30 min at 95°C in Antigen Retrieval Citra Solution, pH 6 (BioGenex Laboratories, Fremont, CA, USA). Sections were incubated with Dual Endogenous Enzyme Block (Dako, Carpinteria, CA, USA) for 30 min, followed by the treatment with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) in Tris-buffered saline (TBS) for 30 min. Samples were then incubated at 4℃ for 16 h with IRS1 antibody (rabbit 06-248, Millipore, Billerica, MA, USA; 1:200 dilution) or IRS2 antibody (rabbit 06-506, Millipore; 1:500). After washing thoroughly in TBS, sections were incubated with anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for 30 min, then treated with streptavidin-peroxidase (ABC kit, Vector Laboratories) according to the manufacturer’s instructions. Specimens were visualized using diaminobenzidine (Dako) and counterstained with hematoxylin. Sections processed with the replacement of primary antibody by TBS were used as a negative control.

Immunohistochemical assessment for IRS1 and IRS2 in all cases were interpreted by a pathologist (T.M.), and a random group of 76 cases was independently reviewed by a second pathologist (S.A.K.). Both pathologists were blinded to any information concerning the CRC cases. Concordance between the two pathologists indicated substantial agreement for both IRS1 status (four levels) with a weighted κ of 0.74 (95% CI 0.61 to 0.86) and IRS2 status (four levels) with a weighted κ of 0.77 (95% CI 0.65 to 0.89). Tumor cytoplasmic IRS1 and IRS2 expression status were scored as 1 (no or minimal staining), 2 (weak staining), 3 (moderately intense staining), and 4 (intense staining) based on the staining intensity in colorectal carcinoma cells.

Statistical analysis

We calculated person-years for each participant from 1988 when the shift work questionnaire was returned, to the date of death, CRC diagnosis, or the end of follow-up (June 1, 2012), whichever came first. We used the duplication-method Cox proportional hazards regression for competing risks data to calculate age-adjusted and multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for each CRC subtype.(34) Multivariable hazard ratios were adjusted for age, BMI, smoking, history of colorectal cancer in a parent or sibling, history of sigmoidoscopy / colonoscopy, postmenopausal status and hormone use, physical activity, regular aspirin use, alcohol consumption, total intake of vitamin D, folate, calcium, red meat and processed meat, sleep duration, and history of type 2 diabetes. For covariates, when appropriate, we have calculated the cumulative averages by averaging all the prior intakes up to each questionnaire cycle. All the models were stratified by age (in months) and year of questionnaire return (every two years since baseline questionnaire return). To retain sufficient statistical power in the analysis, we divided the duration of rotating shift work into three categories as never, 1 – 14, and ≥ 15 years as main exposure. When appropriate, we calculated cumulative averages for covariates including consumptions of alcohol, vitamin D, folate, calcium, red meat and processed meat. For each covariate with missing data (generally 2 – 3%), we assigned a separate “missing” indicator to include those participants in the multivariable Cox models. We found no violation of the proportional hazard assumption.

To retain statistical power in subgroup analysis, tumors were classified as IRS1- or IRS2-positive (moderate / intense) and IRS1- or IRS2-negative (negative / weak) with the score ranging from 3 to 4 and 1 to 2, respectively. We examined the statistical significance of the difference in associations according to cancer subtypes using the likelihood ratio test that compared the model fit that allowed separate associations by different tumor IRS1 or IRS2 expression status with the model fit that assumed a common effect. Linear trend tests were conducted using the median of each category of night-shift work duration as a continuous variable, and the P value for trend was calculated using a Wald test. We also conducted a sensitivity analysis using inverse probability weighting method as previously described to reduce the potential bias due to the availability of tumor samples.(34,35) We used duplication-method Cox regression cause-specific hazards regression for competing risk data (34) to assess the association between night-shift work and risks of colorectal cancer subtypes by IRS1 or IRS2. In this competing risk model, the outcome of interest is the incidence of a specific subtype of CRC, but not CRC, and therefore, cases without the specific biomarker data were treated as censored as the diagnosis of colorectal cancer. The weight was set as the reciprocal of the predictive probability for each case with the corresponding IRS1 or IRS2 marker, whereas, it was set as 1 for non-cases or cases without the corresponding IRS1 or IRS2 marker in the weighted Cox regression models.

We did a secondary data analysis stratified by primary tumor location (colon vs. rectum). All analyses were performed using the SAS software (SAS Institute, Version 9.2, Cary, NC), and a two-sided P value less than 0.05 was considered statistically significant for the overall risk testing. For subtype analysis, the primary hypothesis test was the heterogeneity in the association with various colorectal cancer subtypes. To account for multiple testing for two biomarkers (IRS1 and IRS2), we adjusted for the statistical significance level to 0.025 (0.05/2).

RESULTS

Among 77,470 eligible participants reporting their night-shift work history in 1988 with 1,708,790 person-years of follow up, we documented a total of 1,397 incident colorectal cancer cases, of which 304 or 308 had available IRS1 or IRS2 expression data respectively (Figure 1). Compared with women who never worked rotating night-shifts, women with longer duration of rotating night-shift work were more likely to be a smoker, overweight, sleepless, and developing type 2 diabetes (Table 1). Additionally, demographic or clinical features were similar according to availability of tumor IRS1 or IRS2 status (Supplementary Table S1). Among the CRC cases with available tissue for IRS1 or IRS2 expression analysis, 86 (28.2%) and 102 (33.1%) had moderate or intense IRS1 and IRS2 expression, respectively.

Table 1.

Age-adjusted baseline characteristics of participants by night-shift work duration in the Nurses’ Health Study (Women, at 1988)

	Night-shift work duration
Characteristic	Never	1 - 14 years	≥ 15 years
N of participants	31,382	40,359	5,729
Age, years*	54.7 (7.2)	54.7 (7.1)	54.8 (7.1)
Race (White), %	97.9	97.5	96.5
Body mass index, kg/m2	25.3 (4.8)	25.6 (4.9)	26.9 (5.5)
Family history of colorectal cancer, %	11.4	11.6	12.1
History of sigmoidoscopy / endoscopy, %	12.4	12.6	11.4
Postmenopausal status, %	72.1	72.5	76.0
Postmenopausal hormone use, %	37.5	38.1	35.5
Total activity, METS - hours/week§	14.6 (20.8)	16.0 (22.0)	16.6 (24.0)
Regular aspirin use (2 or more tablets/week), %	39.3	40.9	42.6
Smoking status, %
Never, %	45.6	43.3	42.2
Smoking, pack years 0 −10, %	16.8	17.4	14.4
Pack years > 10, %	36.3	37.9	41.7
Total alcohol intake, g/day	6.2 (10.6)	6.3 (10.7)	5.2 (10.3)
Total vitamin D, IU/day	341 (252)	343 (253)	337 (255)
Total folate intake, μg/day	403 (221)	407 (224)	395 (218)
Total energy intake, kcal/day	1747 (519)	1782 (525)	1789 (556)
Red meat, servings/week	2.2 (1.4)	2.2 (1.4)	2.2 (1.4)
Processed meat, servings/week	1.0 (1.3)	1.0 (1.3)	1.1 (1.3)
Total calcium intake, mg/day	1093 (514)	1088 (507)	1056 (508)
Sleep duration, %
Sleep < 6h, %	3.1	3.8	8.4
Sleep 6h -< 7h, %	20.5	22.4	28.2
Sleep 7h -< 8h, %	50.2	49.5	44.0
Sleep 8h -< 9h, %	21.9	20.3	15.7
Sleep ≥ 9h, %	4.3	3.9	3.6
History of type 2 diabetes, %	4.1	4.3	6.7

Values were means ± standard deviation (SD) or percentages and were standardized to the age distribution of the study population.

^*Value was not age adjusted.

^§METS, metabolic equivalent task score.

Consistent with our previous report,(4) we observed a trend of increased overall risk of CRC with at least 15 years of night-shift work (P_trend = 0.06). Additionally, this positive association appeared to persist when we restricted our analyses to women with available IRS1 or IRS2 expression data (Table 2). We also examined the association between night-shift work duration and CRC risk by primary tumor sites. We found a similar significant trend of increasing risk of rectal cancer (15+years vs. never: multivariable HR=1.54; 95%CI, 1.03 to 2.29) as in Papantoniou et al (same comparison, multivariable HR=1.60; 95%CI, 1.09 to 2.34).

Table 2.

Night-shift work duration and colorectal cancer risk according to tumor IRS1 and IRS2 expression status in the Nurses’ Health Study

	Night-shift work duration
	Never	1 - 14 years	≥ 15 years	Ptrend*	Pheterogeneity¶
Total colorectal cancer in the full cohorts
No. cases (N = 1,397)	536	718	143
Age-adjusted HR (95% CI)	1 (ref)	1.01 (0.90 to 1.13)	1.28 (1.06 to 1.54)	0.008
Multivariable HR (95% CI)§	1 (ref)	1.01 (0.90 to 1.13)	1.20 (0.99 to 1.45)	0.06
Total colorectal cancer among women with IRS1 data
No. cases (N = 304)	122	146	36
Age-adjusted HR (95% CI)	1 (ref)	0.91 (0.71 to 1.16)	1.42 (0.97 to 2.06)	0.05
Multivariable HR (95% CI)§	1 (ref)	0.90 (0.70 to 1.14)	1.31 (0.89 to 1.91)	0.13
IRS1
Negative / weak
No. cases (N = 218)	90	105	23
Age-adjusted HR (95% CI)	1 (ref)	0.88 (0.66 to 1.17)	1.23 (0.77 to 1.94)	0.36	0.02
Multivariable HR (95% CI)§	1 (ref)	0.87 (0.65 to 1.15)	1.13 (0.71 to 1.80)	0.56	0.02
Moderate / intense
No. cases (N = 86)	32	41	13
Age-adjusted HR (95% CI)	1 (ref)	0.98 (0.62 to 1.56)	1.96 (1.02 to 3.77)	0.03
Multivariable HR (95% CI)§	1 (ref)	0.97 (0.61 to 1.54)	1.81 (0.94 to 3.48)	0.06
Total colorectal cancer among women with IRS2 data
No. cases (N = 308)	119	153	36
Age-adjusted HR (95% CI)	1 (ref)	0.98 (0.77 to 1.25)	1.46 (1.00 to 2.13)	0.04
Multivariable HR (95% CI)§	1 (ref)	0.97 (0.76 to 1.23)	1.35 (0.92 to 1.97)	0.11
IRS2
Negative / weak
No. cases (N = 206)	90	98	18
Age-adjusted HR (95% CI)	1 (ref)	0.83 (0.62 to 1.11)	0.98 (0.59 to 1.63)	0.95	0.008
Multivariable HR (95% CI)§	1 (ref)	0.83 (0.62 to 1.10)	0.90 (0.54 to 1.51)	0.72	0.008
Moderate / intense
No. cases (N = 102)	29	55	18
Age-adjusted HR (95% CI)	1 (ref)	1.46 (0.93 to 2.29)	2.92 (1.61 to 5.30)	0.0004
Multivariable HR (95% CI)§	1 (ref)	1.42 (0.90 to 2.22)	2.69 (1.48 to 4.89)	0.001

CI, confidence interval; HR, hazard ratio.

Duplication-method Cox proportional cause-specific hazards regression for competing risks data was used to compute HRs and 95% CIs.

All analyses were stratified by age (in month) and year of questionnaire return.

^*Linear trend test using the median years of each category.

^¶The likelihood ratio test was used to test for the heterogeneity of the associations between night-shift work duration (median) and colorectal cancer risk according to the expression of IRS1 and IRS2 (ordinal).

^§Multivariable hazard ratios were adjusted for age (in month), adult BMI (< 25, 25 -< 27.5, 27.5 -< 30, or ≥ 30 kg/m²), smoking (0, 1-10, or > 10 pack-years), history of colorectal cancer in a parent or sibling (yes or no), history of sigmoidoscopy/colonoscopy (yes or no), postmenopausal status and hormone use (premenopause, postmenopause and never use hormone, postmenopause and current use hormone, postmenopause and past use hormone), physical activity (< 3, 3 -< 27, ≥ 27 METS - hours/week), regular aspirin use (yes or no), alcohol consumption (0 -< 5, 5 -< 15, or ≥ 15 g/day), total intake of vitamin D, folate, calcium, red meat and processed meat (all in tertiles), sleep duration (< 6h, 6 -< 7h, 7 -< 8h, 8 -< 9h, or ≥ 9h), and history of type 2 diabetes (yes or no).

We then tested our primary hypothesis that the association between duration of rotating night-shift work and CRC risk might differ according to IRS1 or IRS2 expression. We found that the positive association of longer duration of rotating night-shift work appeared to differ by tumoral IRS1 or IRS2 status. Compared with women who never worked rotating night-shifts, women with at least 15 years of rotating night-shift work had a trend of an increased risk for IRS1-positive tumors (multivariable HR = 1.81, 95%CI 0.94 to 3.48, P_trend = 0.06), but not for IRS1-negative tumors (multivariable HR = 1.13, 95%CI 0.71 to 1.80, P_trend = 0.56, P_{heterogeneity} for IRS1 subtypes = 0.02). Likewise, a stronger association was observed for the IRS2-positive tumors (multivariable HR = 2.69, 95% CI 1.48 to 4.89, P_trend = 0.001) but not for the IRS2-negative tumors (multivariable HR = 0.90, 95% CI 0.54 to 1.51, P_trend = 0.72, P_{heterogeneity} for IRS2 subtypes = 0.008) (Table 2).

In order to reduce possible bias due to the availability of tumor specimens after diagnosis of CRC, we conducted a sensitivity analysis using the inverse probability weighting (IPW) method as previously described. We observed similar differential associations by both IRS1 (P_{heterogeneity} = 0.001) and IRS2 status (P_{heterogeneity} = 0.001) (Supplementary Table S2). In addition, the similar pattern was observed regardless of tumor locations in either colon or rectum, although the heterogeneity test did not reach statistical significance (Supplementary Table S3).

DISCUSSION

As the third most commonly diagnosed cancer both in women and men in US and worldwide,(36,37) CRC comprises a group of heterogeneous diseases in which each tumor arises and behaves in a unique fashion due to its distinctive genetic and epigenetic background. The potential pro-tumorigenic effects of night-shift work on CRC may thus differ by specific tumor molecular subtypes. In this large U.S. prospective cohort of nurses, we found that working a rotating night-shift for at least 15 years was associated with higher risk of IRS2-positive and had a trend of higher risk of IRS1-positive CRCs, but not negative tumors, compared with women who never worked rotating night-shifts.

Consistent with previous studies including Schernhammer’s in 2003,(3–5) we observed positive associations between rotating night-shift work and CRC risk. However, Papantoniou et al published an updated analysis of Schernhammer et al of night shift work and CRC in NHS, and newly adding data from the NHS2 cohort,(9) which we did not include in the current paper due to lack of tumor marker data in NHS2. Our findings regarding to overall association of night-shift work duration (i.e., 15+ years night shift work vs. never) and CRC risk in the NHS cohort (multivariable HR=1.20, 95%CI: 0.99 to 1.45) are consistent with these results (multivariable HR=1.15, 95%CI: 0.95 to 1.39). There were also some differences in the inclusion criteria and covariate adjustment between these two studies. Specifically, Papantoniou included 130 additional CRC cases (N=1,527) compared to ours (N=1,397), as these 130 CRC cases were histologic subtypes of malignancies (carcinoid, leiomyosarcoma, and squamous cell cancer), which may have different pathogenesis and were therefore not suitable for MPE analysis. Lastly, we adjusted for additional covariates in our paper that were not included in Papantoniou’s paper, all of which likely contributed to the slight difference in magnitude of the aforementioned associations.

Working at night and rotating shifts could lead to a series of unfavorable alterations of the sleep cycle and cell cycle,(38) lipid and carbohydrate metabolism, and insulin resistance.(39,40) Because these alterations play a role in regulation of cell proliferation, the observed positive association is biological plausible.(1,41) The insulin resistance system involving the insulin receptor (INSR) and IGF1R pathways is the primary system responsible for many manifestations of metabolic disorders. Insulin is also considered as a growth factor for tumor formation by stimulating proliferation, inhibiting apoptosis or activating the INSR and IGF1R pathway.(42) Recent population studies showed that circulation IGF1 and IGF2, and some of genetic variants in the INSR or IGF1R pathway (such as single-nucleotide polymorphisms in IGF1, IGFBP3, INSR and IRS), were associated with CRC risk. (43–48) Therefore, the influence of night-shift working on colorectal cancer might partially act through the INSR and IGF1R pathway. The positive associations of CRC risk and night-shift work observed for IRS2-positive tumors, and the marginally significant association for IRS1-positive tumors support this possibility.

IRS proteins are a family of cytoplasmic proteins composed of six members (IRS1 to 6) that regulate numerous processes such as growth, metabolism, survival and proliferation.(49) IRS1 and IRS2 were identified as the first two dominant members of the IRS family, which act as the mediators of the INSR and IGF1R pathway and play a central role in maintaining diverse cellular functions, such as metabolism and proliferation.(24,50) In normal metabolic regulation, these proteins contribute to the insulin-regulated glucose homeostasis through promoting glucose uptake and utilization, and regulating the biosynthesis of macromolecules that are required for cell growth and proliferation.(50) When human circadian rhythms are disrupted, such as in night-shift workers, glucose homeostasis is dysregulated, leading to hyperinsulinemia and insulin insensitivity, as well as potentially insulin resistance. In vitro, high levels of insulin may stimulate IRS1 tyrosine phosphorylation, which is associated with activation of PI3K / AKT and MAPK pathway, and mitogenesis in mouse skeletal muscle cells.(19) Similarly, chronic insulin exposure may be associated with IRS1 and IRS2 expression, AKT activation and chemo-resistance in some colon cancer cells.(18) Phosphorylation of IRS1 tyrosine sites could activate downstream pathways including PI3K / AKT, MAPK, and PAK1, which increase proliferation and cell survival in cancer cell.(51,52) Many studies have focused on the increased expression level or activity of IRSs in different human cancers including colorectal cancer, and correlated these with poor prognosis, potentially defining them as oncogenic proteins.(23,53) In light of this evidence, night-shift workers may experience different degrees of metabolic disorders such as insulin oscillations or hyperinsulinemia, which can stimulate IRSs and its downstream signaling. This disruption can eventually result in tumor occurrence as the duration of exposure (i.e., night-shift work) increases. Hence, it is plausible that the higher risk of longer duration of shift work appeared in IRSs-positive CRC but not IRSs-negative tumors.

We also observed slightly stronger positive associations with IRS2-positive than IRS1-positive tumors, suggesting a possible different role of IRS1 and IRS2 in tumorigenesis. To date, most such research has focused on breast cancer. Using the PyV-MT mouse model of mammary tumor progression, it was reported that tumor onset and growth were equivalent in the absence of either IRS1 or IRS2.(54,55) However, the absence of IRS2 was associated with the regression of mammary tumor metastasis but IRS1 cannot compensate for this loss.(55) And in irs1−/− tumors, IRS2 activation was enhanced and associated with a higher frequency of metastasis.(54) Moreover, IRS1 was expressed predominantly in estrogen receptor positive, well-differentiated breast cancer cell lines, whereas IRS2 was expressed in estrogen receptor negative, poorly-differentiated metastatic breast cancer cells.(56,57) Taken together, these studies suggest that IRS2 might play a different role than IRS1 in tumor initiation, aggressiveness, and progression. Further functional studies in colon cancer pre-clinical models are warranted to further clarify these potential biological mechanisms.

Our study has several strengths, including the prospective design with a large sample size, long-term follow-up with high follow-up rate, and validated CRC outcomes. The repeated assessments of a variety of dietary and lifestyle risk factors allowed better confounding control. Furthermore, the availability of tumor IRS1 and IRS2 data in these cohorts enabled us to identify tumor subtypes that are more susceptible to night-shift work, which provide potential mechanistic insights.

Our study has some potential limitations. First, information on lifetime shift work exposure was self-reported, and only inquire once with no further updates beyond 1988. We are unable to evaluate the impact of changes or different intensities or patterns of night-shift work. However, it is likely that these self-reported data among these nurses were reliable because other self-reported measures by these nurses have been reasonably accurate.(4) Second, not all colorectal cancer cases in the NHS cohort have tumor specimen data from which we can assess their IRS status. However, patients with or without IRS data were highly comparable. Additionally, to address possible bias due to the availability of tumor specimens, we used IPW in sensitivity analyses and results remained essentially unchanged. Nonetheless, the number of cases with IRS data in our study was limited and chance can therefore not be ruled out. Finally, due to sparse data in certain tumor subtype analyses especially in the long-term shift worker group, we could not have enough power to analyze these association by stratify or adjust for other potential confounding molecular features. So, these results should be interpreted cautiously.

In conclusion, our prospective cohort study showed that working at least 15 years of rotating night-shift was associated with higher risk of CRC, particularly for IRS2-positive tumors, and with a trend for higher risk of IRS1-positive CRCs with increasing duration of night shift work. Our findings suggest a role of IRSs, especially for IRS2, in mediating pro-tumorigenic effects of night-shift work on CRC. Future studies with more available tumor specimens and functional experiments are needed to confirm these findings and better clarify the underlying mechanisms.

Abbreviations:

CI

confidence interval

CRC

colorectal cancer

FFPE

formalin-fixed paraffin-embedded

FFQ

food frequency questionnaire

HR

hazard ratio

IGF1R

insulin like growth factor 1 receptor

INSR

insulin receptor

IRS

insulin receptor substrate

METS

metabolic equivalent task score

MPE

molecular pathological epidemiology

NHS

Nurses’ Health Study

TBS

Tris-buffered saline

TMA

tissue microarrays