Abstract
Background
Current and recent smoking have been associated with a greater risk of prostate cancer recurrence and mortality, though the underlying mechanism is unknown.
Methods
To determine if telomere shortening, which has been associated with poor outcomes, may be a potential underlying mechanism, we prospectively evaluated the association between smoking status and telomere length in 567 participants in the Health Professionals Follow-up Study, who were surgically treated for prostate cancer. Using tissue microarrays (TMA), we measured telomere length in cancer and benign tissue, specifically stromal cells in the same TMA spot using a telomere-specific fluorescence in situ hybridization assay. Smoking status was collected via questionnaire 2-years before diagnosis. Adjusting for age, pathologic stage and grade, the median and standard deviation of the per-cell telomere signals were determined for each man for stromal cells and cancer cells by smoking categories. In sub-analyses, we restricted to men without major co-morbidities diagnosed before prostate cancer.
Results
Overall, there were no associations between smoking status and telomere length or variability in stromal cells or cancer cells. However, among men without comorbidities, current smokers and former smokers who quit <10 years ago had the most variable telomere length in stromal cells (29.3% more variable than never smokers; p-trend=0.0005) and in cancer cells (27.7% more variable than never smokers; p-trend=0.05). Among men without comorbidities, mean telomere length did not differ by smoking status in stromal cells or cancer cells.
Conclusion
Telomere variability in prostate cells may be one mechanism through which smoking influences poor prostate cancer outcomes.
Keywords: Smoking, Telomere Length, Prostate Cancer, Stromal Cells
Introduction
Current or recent cigarette smoking has been associated with an increased risk of advanced stage and high grade prostate cancer at diagnosis, prostate cancer progression independent of stage and grade, and prostate cancer mortality, but not prostate cancer incidence1, 2. The biologic mechanism(s) underlying these associations have not been established, though one possible mechanism may be through alterations in telomere length.
Telomeres, which protect the chromosomes from degradation and recombination, can become dysfunctional when there is incomplete replication during DNA synthesis, alterations of telomere-binding proteins involved in telomere maintenance, or DNA damage via oxidative stress3–5. Though cancer cells, including prostate cancer cells6–8, typically have significantly shorter telomeres than normal cells from the same tissue, cancer cell maintenance of telomeres improves viability9. Among cancer cells, variability in telomere length may indicate more genetic instability, and thus could promote the development of a more aggressive phenotype10. We recently reported that, among men surgically treated for prostate cancer, those with shorter telomeres in prostate cancer associated stromal cells (i.e., stromal cells in the same tissue microarray [TMA] spot as cancer) and those with more variability in telomere length among prostate cancer cells had a higher risk of poor prostate cancer outcomes, in particular prostate cancer death11.
Cigarette smoking, a source of oxidative stress, has been associated with shorter telomere length in peripheral blood leukocytes in some large, high quality studies (e.g.12). However, the influence of cigarette smoking on telomere length in prostate cells is unknown. Therefore, we conducted a prospective analysis of the association between cigarette smoking and telomere length in 567 men surgically treated for clinically-localized prostate cancer, who were participants in the Health Professionals Follow-up Study. We measured telomere length using a telomere-specific fluorescence in situ hybridization (FISH) assay that provides single cell resolution of telomere length while maintaining tissue architecture. We hypothesized that men who currently smoked or quit within the past decade would have shorter prostate cell telomere length as compared with men who never smoked, especially in stromal cells, whereas in cancer cells we did not expect associations between smoking status and telomere length or telomere length variability.
Materials and Methods
Study Population
We conducted a prospective study of 567 men surgically treated for prostate cancer (median year of diagnosis: 1994) who participated in the Health Professionals Follow-up Study (HPFS)11. The HPFS began in 1986 with 51,529 men, aged 40-75 years, who completed a mailed questionnaire on demographics, lifestyle factors and medical history. The men have been asked to complete questionnaires every two years since baseline; response among men eligible to receive them is 94%. The conduct of the HPFS was approved by the Human Subjects Committee of the Harvard School of Public Health. The study on telomere length in prostate tissue was additionally approved by the Institutional Review Board at the Johns Hopkins Bloomberg School of Public Health.
Measures of Smoking Status
At baseline, men reported their current smoking status, past smoking status, time since quitting, and the average number of cigarettes smoked per day before age 15 years, ages 15 through 19, 20 through 29, 30 through 39, 40 through 49, 50 through 59, and 60 years and older. Pack-years was calculated as years of smoking multiplied by the average number of packs (1 pack: 20 cigarettes) smoked per day. Men reported updated smoking status every 2 years. To determine current smoking status, we selected the smoking status reported on the questionnaire immediately preceding the year in which the man was diagnosed with prostate cancer. Men were categorized into never smokers (50%), former smokers who quit ≥10 years ago (32%), and current smokers/former smokers who quit <10 years ago (18%). Because cigarette smoking can influence many co-morbid conditions that we hypothesize may also influence telomere length, we conducted sub-analyses among men who had no major co-morbid conditions prior to prostate cancer diagnosis. With respect to comorbidities, on the baseline questionnaire, and on each follow-up questionnaire, men reported whether they had the following conditions professionally diagnosed: high blood pressure, elevated cholesterol, diabetes, and myocardial infarction (heart attack). We categorized men who answered no to all of these conditions on each of the 1986, 1988 and 1990 questionnaires (e.g. the questionnaires prior to prostate cancer diagnosis) as men without a major comorbid condition diagnosed before prostate cancer (n=271; never smokers: 51%, former smokers who quit ≥10 years ago: 29%, and current/recent smokers: 20%).
Additionally, because there appears to be an interaction between smoking and vitamin E use in the association with fatal prostate cancer such that vitamin E use offsets the association between smoking and fatal prostate cancer13, we performed a sub-analysis restricted to men not taking supplemental vitamin E (Vitamin E ≤15 mg; n=350; never smokers: 49%, former smokers who quit ≥10 years ago: 32%, and current/recent smokers: 19%).
Tissue Collection and Tissue Microarray Construction
The ascertainment of prostate cancer in the HPFS has been described previously11. With participant permission, tissue blocks of the prostatectomy specimens were obtained from the original pathology departments. H&E-stained tissue sections were re-reviewed by study pathologists and assigned a standardized Gleason sum14. For this project, we used five tissue microarrays (TMAs) that had been constructed11, sampling at least three areas of the tumor focus that was the largest and/or had the highest Gleason sum.
Measurement of Telomere Length
Tissue microarray sections containing areas of adenocarcinoma and benign tissue were stained using a telomere-specific fluorescence in situ hybridization (FISH) probe and 4′,6-diamidino-2-phenylindole (DAPI) for labeling total nuclear DNA11. Image analysis was used to quantify telomeric signals in individual cancer cells, and in these same TMA spots with cancer, stromal cells (lymphocytes excluded), basal epithelial cells and luminal epithelial cells in non-cancer areas. For each cell type, 30 to 50 individual cells per man were analyzed, but not all cell types were available for evaluation for some men11.
Statistical Analysis
Means and proportions for demographic and other factors by smoking categories were calculated; differences across smoking categories were evaluated using Wald test from regression and chi-square tests, respectively. Median (telomere length) and standard deviation (telomere length variability) of the telomere signal normalized to DAPI were determined for each man for cancer cells and non-cancer cells by categories of smoking. We evaluated the associations between smoking and telomere length and variability in telomere length using linear regression. All analyses were adjusted the potential confounders: age at diagnosis (continuous), and known prognostic factors, prostatectomy Gleason sum (categorical: ≤6, 3+4, 4+3, ≥8), and pathologic TNM stage (categorical ≥T3b, or N1, or M1). Additional adjustment for body mass index and physical activity level was performed; associations were not appreciably different and thus the results presented are not adjusted for body mass index and physical activity level. In sub-analyses, we restricted to men (1) without major comorbid conditions diagnosed before prostate cancer and (2) not taking supplemental vitamin E. All analyses were performed using SAS v 9.2 (SAS Institute, Cary, NC). All statistical tests were two-sided, with P<0.05 considered to be statistically significant.
Results
Men who currently smoked or quit smoking within the prior 10 years (subsequently referred to as ‘current/recent’ smokers) were similar to men who had quit smoking 10 or more years prior (subsequently referred to as ‘former’ smokers) and men who had never smoked on most characteristics (Table 1). Former smokers were older than current/recent and never smokers. Compared with former smokers, current/recent smokers accumulated significantly more pack years. Compared with former and never smokers, current/recent smokers were significantly more likely to have a Gleason sum of 4+3 or higher, and pathologic stage T3b or higher, though this difference in stage was not statistically significant.
Table 1.
Characteristics of men surgically treated for clinically localized prostate cancer by pre-diagnostic* smoking status, HPFS.
| Smoking Status
|
||||
|---|---|---|---|---|
| Never (n=281) |
Former, quit ≥10 years ago (n=184) |
Current and Former, quit <10 years ago (n=102) |
P-trend** | |
| Mean age at diagnosis, years (std) | 64.7 ± 6.3 | 66.7 ± 6.2 | 64.1 ± 5.4 | 0.0003 |
| White (%) | 95.4 | 95.7 | 94.1 | 0.83 |
| Mean Pack-years (std) | – | 18.7 ± 13.9 | 41.1 ± 19.6 | <0.0001 |
| Comorbidities (%) | ||||
| High Blood Pressure | 27.1 | 32.6 | 25.5 | 0.32 |
| Diabetes | 3.2 | 2.2 | 4.9 | 0.45 |
| High Cholesterol | 33.8 | 32.6 | 25.5 | 0.30 |
| Myocardial Infarction | 3.6 | 6.0 | 7.8 | 0.20 |
| Any Comorbidity | 51.3 | 57.1 | 46.1 | 0.18 |
| Gleason sum (%) | ||||
| ≤6 | 21.4 | 26.1 | 15.7 | |
| 3+4 | 37.0 | 39.1 | 27.5 | 0.02 |
| 4+3 | 23.8 | 18.5 | 36.3 | |
| ≥8 | 17.8 | 16.3 | 20.6 | |
| Pathologic stage ≥T3b (%) | 11.7 | 10.9 | 18.6 | 0.13 |
| Mean PSA at diagnosis in ng/mL (std) | 11.5 ± 14.4 | 10.3 ± 10.5 | 10.5 ± 10.1 | 0.60 |
| % Missing | 11.7 | 13.6 | 16.7 | |
Pre-diagnostic mseasures collected within 2 years prior to diagnosis;
Wald test from regression (means), Chi-square test (proportion)
Stromal Cells, Basal Epithelial Cells and Luminal Epithelial Cells
When all men were categorized by their pre-diagnostic smoking status, there was no difference in telomere length or variability in telomere length in stromal cells (Table 2). Similarly, among men without major pre-diagnostic comorbidities, there was no difference in telomere length in stromal cells. However, among men without major pre-diagnostic comorbidities, current/recent smokers had 29.3% more variability in telomere length in stromal cells as compared to never smokers (P-trend=0.0005; Table 2). The results were similar when restricting to men without the most common co-morbidities (i.e. high blood pressure and high cholesterol; p-trend =0.002). Among men who did not take a vitamin E supplement, there was no significant difference in variability in telomere length in stromal cells as compared to never smokers (P-trend=0.1). There was no difference in telomere length or variability in telomere length by smoking status in basal epithelial cells or in luminal epithelial cells in all men, or in men without co-morbidities (p>0.05).
Table 2.
Association of pre-diagnostic smoking status with prostate stromal cell telomere length in men surgically treated for clinically localized prostate cancer, overall and among men without co-morbidities, HPFS
| N | Adjusted mean telomere length (95% CI) |
Difference in adjusted mean telomere length | Adjusted mean telomere length variability (95% CI) |
Difference in adjusted mean telomere length variability | |
|---|---|---|---|---|---|
| Overall | |||||
| Never | 281 | 59.5 (55.2, 63.8) | Reference | 26.9 (24.0, 29.9) | Reference |
| Former, quit ≥10 years ago | 184 | 57.5 (52.5, 62.5) | −3.4% | 26.8 (23.3, 30.2) | −0.4% |
| Current and Former, quit <10 years ago | 102 | 57.5 (51.4, 63.5) | −3.4% | 29.7 (25.6, 33.8) | +10.4% |
| PTrend | 0.44 | 0.31 | |||
|
| |||||
| No Comorbidities* | |||||
| Never | 138 | 57.4 (51.2, 63.6) | Reference | 24.6 (21.8, 27.3) | Reference |
| Former, quit ≥10 years ago | 79 | 59.1 (50.9, 67.3) | +3.0% | 27.7 (24.1, 31.4) | +12.6% |
| Current and Former, quit <10 years ago | 54 | 59.8 (51.1, 68.5) | +4.2% | 31.8 (27.9, 35.6) | +29.3% |
| PTrend | 0.57 | 0.0005 | |||
Adjusted for age at diagnosis, pathologic stage and grade.
Men with no self-reported, pre-diagnostic diagnoses of high blood pressure, diabetes, high cholesterol, or myocardial infarction.
Cancer Cells
When all men were categorized by their pre-diagnostic smoking status, there was no difference in telomere length or variability in telomere length in cancer cells (Table 3). Similarly, among men without major pre-diagnostic comorbidities, there was no difference in telomere length in cancer cells. However, among men without major pre-diagnostic comorbidities, current/recent smokers had 27.7% more variability in telomere length in cancer cells as compared to never smokers (P-trend=0.05; Table 3). Also, the results were similar when restricting to men without the most common co-morbidities (i.e. high blood pressure or high cholesterol; p-trend =0.05). Among men who did not take a vitamin E supplement, there was no significant difference in variability in telomere length in cancer cells by smoking status (P-trend=0.31).
Table 3.
Association of pre-diagnostic smoking status with prostate cancer cell telomere length in men surgically treated for clinically localized prostate cancer, overall and among men without co-morbidities, HPFS
| N | Adjusted mean telomere length (95% CI) |
Difference in adjusted mean telomere length | Adjusted mean telomere length variability (95% CI) |
Difference in adjusted mean telomere length variability | |
|---|---|---|---|---|---|
| Overall | |||||
| Never | 281 | 16.8 (14.8, 18.7) | Reference | 10.8 (9.6, 12.1) | Reference |
| Former, quit ≥10 years ago | 184 | 15.7 (13.4, 18.0) | −6.5% | 10.1 (8.6, 11.6) | −6.5% |
| Current and Former, quit <10 years ago | 102 | 17.7 (14.9, 20.4) | +5.4% | 11.8 (10.0, 13.6) | +9.3% |
| PTrend | 0.80 | 0.56 | |||
|
| |||||
| No Comorbidities* | |||||
| Never | 138 | 16.6 (13.9, 19.2) | Reference | 10.1 (8.5, 11.8) | Reference |
| Former, quit ≥10 years ago | 79 | 16.3 (12.8, 19.8) | −1.8% | 9.9 (7.7, 12.0) | −2.0% |
| Current and Former, quit <10 years ago | 54 | 17.5 (13.8, 21.3) | +5.4% | 12.9 (10.6, 15.2) | +27.7% |
| PTrend | 0.68 | 0.05 | |||
Adjusted for age at diagnosis, pathologic stage and grade.
Men with no self-reported, pre-diagnostic diagnoses of high blood pressure, diabetes, high cholesterol, or myocardial infarction.
Discussion
We evaluated the associations of smoking status with telomere length in specific prostate cell populations in a prospective study of men surgically treated for prostate cancer. Unlike previous studies of peripheral blood leukocytes telomere length (e.g., ref12), we evaluated the influence of smoking status on telomere length in prostate tissue, the target organ. Overall, there was no difference in telomere length or variability in telomere length in prostate stromal, basal epithelial, or luminal epithelial cells or in prostate cancer cells. However, among men without major co-morbidities, current/recent smokers had significantly more variability in telomere length in both prostate stromal cells and prostate cancer cells as compared with long-term former and never smokers.
Cigarette smoking was not associated with telomere length or telomere length variability in prostate cells in men overall. Because cigarette smoking influences several co-morbid conditions that we hypothesize may also influence telomere length, we conducted sub-analyses among men without major comorbidities. In these sub-analyses, we observed increased cell-to-cell telomere length variability in stromal cells among men who currently or recently smoked. Though we no longer observed this pattern in sub-analyses restricting to men who did not take a vitamin E supplement, which has been shown to mitigate the association between smoking and fatal prostate cancer13. In our prior analyses, we did not observe an association between stromal cell telomere length variability and prostate cancer death after adjustment for prognostic factors11, but did observe greater telomere length variability in stromal cells of men who were overweight/obese15. Thus, it is plausible that lifestyle factors, like cigarette smoking and obesity15, have a biologic influence on telomere length variability in stromal cells. It is unknown, however, whether variability in telomere length in prostate stromal cells influences any point in the natural history of prostate carcinogenesis.
Because telomere dysfunction is a dynamic process that both promotes tumorigenesis and can be a consequence of cancer development, it is less clear how cigarette smoking could influence telomere length variability in cancer cells at the point that a tumor is detectable (e.g. the time of telomere measurement in this study). Nevertheless, among men without major comorbidities, we also observed increased cell-to-cell telomere length variability in prostate cancer cells among men who currently or recently smoked. In our prior study, we observed an increased risk of poor prostate cancer outcomes among men surgically treated for prostate cancer who had increased telomere length variability in prostate cancer cells after adjustment for prognostic factors11. It is possible that increased telomere length variability in cancer cells reflects more generalized genomic instability, which has been related to more aggressive features in cancer10, including prostate cancer16, 17. It is unclear whether cigarette smoking contributes directly to increased telomere length variability among cancer cells or if increased variability is marking more aggressive disease among smokers.
Our study, which, to our knowledge, is the first to investigate the influence of cigarette smoking on cell-to-cell telomere length variability in prostate tissue may inform the observation that current or recent cigarette smoking is associated with an increased risk of poor outcomes among men with prostate cancer. However, this study was not large enough to assess telomere length variability as a potential mediator in the association between current or recent cigarette smoking and prostate cancer with a lethal phenotype. Because all of the men in this study had prostate cancer, we do not know whether our results for prostate cell telomere length variability would be similar in a cohort of men at risk for prostate cancer. The smoking status of participants in this study was well characterized. The method used to measure telomere length was validated, state-of-the-art, and provided single cell resolution6, that allowed comparison by each cellular compartment and estimation of cell-to-cell variability in telomere length. In interpreting our findings for variability in telomere length in stromal cells by smoking status, we cannot distinguish between the variability in telomere length being due to variability in length among the same array of stromal cell types among smokers and non-smokers versus a different array of stromal cell types that have inherent differences in telomere length among smokers and non-smokers. We did not observe evidence to support the latter interpretation. Finally, we performed multiple primary tests; the results for smoking and stromal cell variability (p-trend=0.0005) in men without major comorbidities remained statistically significant when using the Bonferroni corrected p-value (p < 0.002) for deeming statistical significance.
The mechanisms that underlie the association between cigarette smoking and poor prostate cancer-specific outcomes are currently unknown. Because cigarette smoking has been associated with shorter leukocyte telomere length in some large, high quality studies (e.g.12), and we previously found that telomere shortening in prostate stromal cells was associated with poor prostate cancer outcomes11, we evaluated the association between cigarette smoking and telomere length and variability in length in prostate cell and prostate cancer cell populations. In summary, we observed no association among cigarette smoking, and telomere length or variability in telomere length in prostate cells or prostate cancer cells overall. Among men with no comorbidities, we did observe increased telomere length variability among prostate stromal cells and prostate cancer cells in men who currently or recently smoked around the time of diagnosis as compared with men who never smoked or quit more than 10 years before diagnosis. The possible influence of cigarette smoking on telomere length variability merits further study.
Acknowledgments
We would like to thank the participants and staff of the HPFS for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The authors assume full responsibility for analyses and interpretation of these data.
Financial Support: DOD W81XWH-05-1-0030, P50 CA58236, CA55075, CA72036, HL35464, CA133891, CA141298, UM1 CA167552, and DOD W81XWH-05-1-0562. CE Joshu, CM Heaphy, and SA Kenfield supported by the Prostate Cancer Foundation. CE Joshu also supported by The Seraph Foundation.
Footnotes
This is the peer reviewed version of the following article: Joshu CE, Peskoe SB, Heaphy CM, Kenfield SA, Mucci LA, Giovannucci EL, Stampfer MJ, Yoon G, Lee TK, Hicks JL, De Marzo AM, Meeker AK, Platz EA. Current or recent smoking is associated with more variable telomere length in prostate stromal cells and prostate cancer cells. Prostate. 2017 Nov 22. [Epub ahead of print] PubMed PMID: 29164645, which has been published in final form at doi: 10.1002/pros.23462. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Conflict of Interest: The authors declare no potential conflicts of interest.
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