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Similar to TNF-alpha, IL6 is a cytokine associated with inflammation, immune function, and injury response (331). IL6 expression is positively associated with obesity (334). The relationship between IL6 and insulin resistance is complex. Many studies have shown that IL6 is associated with increased insulin resistance (331, 334), however at least

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one study demonstrated that IL-6 increases insulin sensitivity in skeletal muscle cells and that IL6 effects on insulin signaling may be tissue specific (356).

IL6 is expressed in normal and neoplastic breast tissue, but there are conflicting reports on whether IL6 levels differ between normal and malignant tissue. In Chavey et al. (335), IL6 was expressed at a significantly higher level in neoplastic compared to normal breast tissue, but in Basolo et al. (357), IL6 was highest in cultures of normal breast

epithelium and lowest in breast tumor cells. Green at al. (336) found no significant difference in IL6 levels between normal and neoplastic cell cultures. Differences may be due to the timing and laboratory methods used to measure IL6 – in Green et al. (336) and Basolo et al. (357) cells were cultured and IL6 levels were measured after the first (336) or third/fourth passages (357), whereas in Chavey et al. (335) IL6 was measured directly in fresh, non- cultured breast tissue.

In vitro studies indicate that IL6 may have pro- and anti-tumorigenic effects

(reviewed by Knupfer et al. (358)). IL6 has been reported to inhibit growth in several ER- positive and ER-negative breast cancer cell lines (359-361). Danforth et al. (359)

demonstrated that IL-6 and estradiol antagonize each other’s growth effects in MCF-7 cells, and that IL6 causes a small decrease in ER expression and an increase in PR expression in MCF-7 cells. Also, IL-6 induced rounding, reduced cell adhesion, and decreased E-cadherin expression in the ZR-75-1-Tx, T47D, and MCF-7 cell lines (361, 362). IL-6 increased migration of T47D cells (360).

The effects of IL6 in cultured surgical breast specimens are not consistent with the reported in vitro effects. IL6 alone was able to activate transcription of ER-alpha in ER- positive primary breast cell cultures (323). IL6 had no effect on proliferation (measured by

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DNA synthesis) in cultured breast tumor cells (357). According to Basolo et al. (357), IL6 stimulated DNA synthesis in cultures of normal breast, but Asgeirsson et al. (361) reported that IL6 inhibited growth (colony size and number) in normal breast cultures. The same study reported that IL6 did not affect adhesion or E-cadherin expression in normal mammary epithelial cultures (361), suggesting that any effects of IL6 to promote cell discohesion seen in cell lines require earlier cancer initiation and/or promotion events.

One potential mechanism by which alterations in IL6 expression or accumulation might be related to breast cancer is interaction between IL6, estrogen, and the estrogen receptor. IL6 can stimulate estrogen synthesis by inducing aromatase activity in fibroblasts cultured from normal and malignant breast tissue (318). Additionally, Chavey et al. (335) reported that IL6 is expressed more commonly in tumors that were ER-negative, PR- negative, and high grade tumors, but other studies (337, 363) found no association between ER or PR status and IL6 expression.

The most well characterized polymorphism in the IL6 gene is the -174 G/C promoter polymorphism. In luciferase reporter assays in HeLa cells, the (AT)8/12 -174C haplotype was associated with lower baseline expression and reduced response to stimulation by

lipopolysaccharide or interleukin-1. Terry et al. (364) tested not only the -174 G/C

polymorphism, but other promoter polymorphisms including -572 G/C, -597 G/A, and a -373 AT repeat polymorphism in both HeLa and ECV40 cells, and showed that changes in IL6 expression level are likely due to a complex haplotype effect, not the single genotype at position -174. Some have reported that the -174G allele is associated with higher circulating IL6 levels (365), but in other studies the -174 genotype was not associated with serum IL6 (366-369). In light of the work by Terry et al. (364), these differing results may depend on

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differing haplotype structures in the different populations in which the studies were conducted.

Several studies have attempted to determine whether the IL6 -174 G/C polymorphism is associated with breast cancer. In a case-control study of women of Austrian or German descent, Hefler et al. (370) the -174C allele was associated with increased breast cancer risk (370). Slattery et al. (371) reported an inverse association between -174 G/C and breast cancer. Gonzalez-Zuloeta Ladd et al. (372) reported a small, non-significant increase in the odds of breast cancer among -174C allele carriers (GC/CC vs. GG), but they also adjusted for several covariates that either do not affect nt -174 genotype or are potentially on the causal pathway between nt -174 genotype and breast cancer risk. In addition to increasing

imprecision of their estimates, they may have induced confounding or attenuated their effect estimates by adjusting for factors in the causal pathway. Unadjusted results were not

reported, and so it is unknown to what extent these results support those of Hefler et al. (370). Finally, Smith et al. (353) and Litovkin et al. (373) reported no association between the nt -174 genotype and breast cancer, but did not reported the corresponding odds ratio or confidence interval.

In a study of women from the southwestern US, Slattery et al. (371) reported

associations for -174 G/C (rs1800795), -572 G/C (rs1800796), and intron 2 G/A (rs2069832). Haplotypes containing the minor alleles for rs1800797, rs1800795, and rs2069832 were associated with lower obesity in non-Hispanic white women, but these same haplotypes were not associated with breast cancer (374).

Studies have also reported conflicting results on the association between the -174 G/C polymorphism and breast cancer prognosis. In an Australian study, the CC genotype was

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associated with poor histological grade, larger tumor size, and lower ER content (375). In the same study, the CC genotype was associated with almost twice the hazard of death compared to the GC or GG genotype, but was not an independent predictor of survival (375). In

contrast, Demichele et al. (376) reported that the -174C allele is associated with improved disease-free survival and overall survival in node-positive breast cancer patients; the data also suggest that patients who are both ER-positive and -174C allele carriers have the best disease-free survival, but statistical evidence for interaction was not significant, probably due to the small number of patient years in the analysis (376).

Although serum IL6 was not associated with breast cancer incidence in a prospective study of elderly subjects (340), serum levels of IL6 were significantly higher among breast cancer patients compared to women with a negative breast biopsy (338). IL6 is produced in many different tissues in the body, including adipose tissue, and so serum IL6 levels may not reflect breast-specific levels of IL6 (331). The in vitro effects of IL6 combined with the demonstrated link to the estrogen-related proliferation pathway provide a plausible link as to how IL6 may be causally related to breast cancer. Since IL6 may have pro- and anti-tumor effects in breast cells, it is hard to predict what effect an IL6 polymorphism will have on breast cancer risk.