The dyslipidemia-associated SNP on the APOA1/C3/A5 gene cluster predicts post-surgery poor outcome in Taiwanese breast cancer patients: a 10-year follow-up study

Breast cancer is currently the leading cause of cancer deaths in females worldwide, and also the second most common cancer after lung cancer [1]. The current therapeutic regimens for operable early-stage breast cancers include endocrine therapy, chemotherapy and radiotherapy. The assessment tools for post-surgery planning are currently based on clinicopathological evaluations, but are unsatisfactory requiring finer adjustments [2]. The risk factors for breast cancer poor prognosis include positive sentinel lymph node metastasis, hormone receptor negativity, larger tumor size, younger age, and menopausal status [2]. In contrast to the Western population, the breast cancer incidence peaks at a younger age in Oriental Asians, which include Taiwanese [3, 4]. Nevertheless, the westernized dietary pattern and lifestyle in Taiwan in the last two decades has increased the incidences of metabolic disorders, including dyslipidemia, as well as breast cancer in Taiwanese females [4].

Both cohort and case–control epidemiological observations have demonstrated the association of metabolic syndrome, obesity and diabetes with increased breast cancer risk [5, 6]. Dyslipidemia often occur in parallel to obesity and diabetes, and is a component of metabolic syndrome [7]. Dyslipidemia in the forms of hypertriglyceridemia, hypercholesterolemia, and low high density lipoprotein-cholesterol (HDL-C) have been observed in breast cancer patients of several ethnic groups [8‐11]. Moreover, in large cohort studies dyslipidemia is associated with increased breast cancer risk and poor prognosis [12‐14]. The intake of lipid-lowering drugs or agents in women was associated with reduced breast cancer occurrence and recurrence risk [15‐17]. Consistently, mammary tumor growth and metastasis was accelerated in a hyperlipidemic murine model [18].

The APOA1/C3/A5 gene cluster transcribes for apolipoproteins (apo) A1, C3 and A5, which regulate HDL-formation and lipoprotein lipase activity [19]. Single-nucleotide polymorphisms (SNPs) on the APOA1/C3/A5 gene cluster are associated with metabolic syndrome, dyslipidemia and diabetes [19]. APOA1 rs670 is associated with altered HDL-C levels, and increased risks of coronary artery disease and metabolic syndrome [20‐25]. APOC3 rs2854116 and rs2854117 are correlated to insulin resistance and non-alcoholic liver steatosis [26]. APOA5 rs662799 and rs2075291 carriers are known to have increased plasma triglyceride [27, 28], and our recent study showed correlation of APOA5 rs662799 with central obesity in males [29]. Though the effects of these SNPs on APOA1/C3/A5 on metabolic disorders have been widely studied and reviewed, their contributions to breast cancer have not been determined in detail.

In this study, we first tested the correlation of 5 selected well-known SNPs on the APOA1/C3/A5 gene cluster with breast cancer in a case–control manner. Furthermore, we analyzed the effect of the SNPs associated with greatest breast cancer risk on patient baseline tumor characteristics, and their post-surgery outcome after a mean follow-up period of 10.4 years. Finally, we specifically examined the prognostic value of APOA1 rs670 in lymph node-negative breast cancer patients.

Dyslipidemia is a known complication of TAM-treatment in breast cancer patients, and increases risk of coronary-artery diseases in patients [33]. Of equal importance, dyslipidemia has been observed in breast cancer patients prior to treatment, and this correlation is supported by epidemiological studies [8‐11]. Concomitantly, usage of lipid-lowering drugs such as statin and niacin has been shown to associate with decreased breast cancer recurrence and risk [15‐17]. SNPs on the APOA1/C3/A5 gene cluster, which is involved in lipid metabolism, are highly associated with dyslipidemia, metabolic syndrome, insulin resistance and responsiveness to relative treatments [19]. However, the contribution of SNPs on the APOA1/C3/A5 gene cluster to breast cancer is yet to be defined. In this study, we tested the effects of dyslipidemia-associated SNPs on the APOA1/C3/A5 gene cluster: APOA1 rs670, APOC3 rs2854116, APOC3 rs2854117, APOA5 rs662799 and APOA5 rs2075291 on breast cancer progression in a Taiwanese patient group with mainly operable early-stage tumors, and a 10-year follow-up interval. We showed that only APOA1 rs670 out of the 5 tested SNPs was correlated to breast cancer, lymph node-positivity, ER/PR double-negativity at baseline. Furthermore, carriers of both minor alleles on APOA1 rs670 had shortest survival time and highest risk in disease progression independent of baseline characteristics. Moreover, the prognostic value of APOA1 rs670 A/A carriage in the worse post-surgery outcomes was most evident in lymph node-negative patients.

The rs670 SNP contains a G-to-A substitution at 75bp upstream of APOA1 transcriptional start site, and is within a MSPI restriction enzyme recognition [19]. A cross-sectional study focusing on the association of APOA1 SNPs with breast cancer and patient phenotype reported that the -75G/A polymorphism correlated with breast cancer risk at baseline [34]. However, the slight discrepancy in associations of -75G/A polymorphism with ER status observed by us and Hamrita et al. is likely due to ethnic (Taiwanese vs. Tunisian) and age differences (62.7% vs. 38.9% <50years) [34]. Nevertheless, in our longitudinal study we found that APOA1 rs670 predicted worse outcome after adjustment for ER/PR status, indicating alternative contributions from APOA1 rs670 in cancer progression. ApoA1 is the structural protein of HDL, and interacts with lecithin cholesterol acyl transferase, which controls the limiting steps in HDL maturation and therefore reverse cholesterol transport [19]. The minor allele of APOA1 rs670 has been correlated to altered HDL-C, diabetes and coronary-artery disease: lower levels of HDL-C was observed in Northern Indians [20], and correlated with severe forms of cardiovascular diseases in Northern Indians, Caucasians in Spain and Australia [20, 24, 25]. Moreover, APOA1 rs670 A/A Spanish carriers had a higher risk of diabetes than their non-A/A counterparts, though their HDL-C did not differ significantly [21, 22]. As plasma apoA1 level is regulated by estrogen and thus TAM treatments, the timing of plasma apoA1 measurement in breast cancer patients is critical. However, we did not have access to TAM treatment-naïve plasma of the patients recruited in this study. Nevertheless, the disease phenotype associated APOA1 rs670 remains significant without HDL-C differences, as has been reported in a number of studies on metabolic/cardiovascular diseases [21, 24, 25].

The significance of plasma HDL-C levels in breast cancer has long been investigated and debated [35]. The current held view is that higher HDL-C levels, which are preventive for cardiovascular events, are also protective for breast cancer. The majority of reports supporting this view were of case–control design, and found lower total or lipid-rich HDL, or higher total cholesterol/HDL-C ratio in breast cancer patients as compared with non-cancerous or other cancer controls in various ethnic groups [8, 10, 11]. A 17.2-year prospective cohort study carried out in Norway found that naive females with the lowest quartile of HDL-C had highest risk of later developing breast cancer [14]. Moreover, the same Norwegian group found the breast cancer patients with highest total cholesterol or lowest plasma HDL-C levels had highest risk in mortality [13]. The female breast is an active site of both lipid uptake and lipid secretion during periods of milk secretion, thus it is readily acceptable that apoA1 is present in human milk [36]. Supportively, apoA1 was also identified in the breast cancer tissues and ex vivo cultured medium. ApoA1 was found in breast tumors, and its amount was correlated positively with chemotherapy resistance in malignant tumors [37]. In contrast, breast tumors later found responsive to chemotherapy secreted higher amount of apoA1 than tumors non-responsive to chemotherapy during short term ex vivo culture, while non-cancerous tissues secreted highest amount of apoA1 [38]. Furthermore, a successful reduction of breast cancer growth in mice by vitamin D treatment was accompanied by decrease of apoA1 production in tumor tissues [39]. It is at this stage unknown how much apoA1 in circulation is contributed by the mammary tissue, but the retention of apoA1 in breast cancer tissues is in accordance with the observation of lower plasma HDL/apoA1 in breast cancer patients. Though the association of plasma lipid profiles and breast cancer incidence or progression is strongly and continuously suggested, the molecular mechanisms of HDL or apoA1 in promoting breast cancer risk are still unclear.

Plasma apoA1/lipid-poor HDL bind to ATP-binding cassette (ABC) lipid transporters during maturation, and the intracellular cholesterol is exported to load onto HDL, facilitating reverse cholesterol transport [40]. Several members of the ABC family were found in mammary tissues, but only those associated with drug resistance were increased in breast cancers as compared with normal tissues [41]. In contrast, ABC transporters involved in lipid transport, including ABC-A1 and ABC-A3, were decreased in breast cancer tissues as compared with healthy mammary glands [42]. In vitro studies found that over-expression of ABC-G1 in breast cancer cell line induces cellular cholesterol efflux and apoptosis [43]. Moreover, ABCA- or ABCG-HDL binding concomitantly induces intracellular signaling and suppresses inflammatory reactions, which is known to be a key factor in tumorigenesis. Binding of ABCA or ABCG by apoA1 induces the recruitment of phosphrylated STAT-3 and JAK2, and thus suppresses the inflammatory pathways including TL4-signalling [40]. Furthermore, ABCA induces Cdc42 and PAK-1 signaling, which in turn are regulative for breast cancer ER/PR expression in vitro and in vivo[44, 45]. Therefore, the altered HDL/apoA1 levels observed in APOA1 rs670 A carriers may participate in the tumorigenesis, survival, ER/PR status of breast cancer cells. In a previous published study, we found that genetic and intra-tumoral ER polymorphisms were correlated with breast cancer in Taiwanese females [30, 31]. It would be of interest how APOA1 rs670 and HDL-C interact with ER polymorphisms both at a systemic and local (mammary tissue) level in these patients.

Clinically, our finding of worst outcome in APOA1 rs670 A/A breast cancer patients as compared to their non-A/A counterparts provides pivotal implications in personalized treatment regimen and therapeutic strategies. The median recurrence-free and overall survival years of APOA1 rs670 A/A carrying breast cancer patients were both longer than the standard chemotherapy duration (~1 year) and the advised 5-year limit for continuous TAM usage. As the patients recruited in this study were non-obese and of early tumor-staging, the administration of adjuvant therapy may be less intensive. Furthermore, the prognostic value of APOA1 rs670 was even more evident in the lymph node-negative patients, who were given less adjuvant therapies under the current assessment guidelines. Thus APOA1 rs670 A/A carriers may benefit from advanced therapies. Recent studies demonstrated that administration of apoA1 mimetic peptides reduced the development of cancer in murine models [46, 47]. Furthermore, a large cohort study on the effect of post-diagnosis statin use in breast cancer females reported a lowered risk in cancer recurrence [48]. These reports suggest that raising HDL-C or apoA1 may be preventive or therapeutic for breast cancers, especially in the APOA1 rs670 A/A carrying patients. On the other hand, due to the low plasma HDL-C and tumor ER/PR negativity in the APOA1 rs670 A/A carriers, the usage of TAM in these breast cancer patients may be controversial and require further evaluation.

MCH, WCH, and KCY conceived and designed the study. MCH and KCY developed the methodology, performed laboratory experiments, and supervised the study. MCH, WCH, and KTL acquired and prepared clinical data. MCH, WCH, KTL, CHW, HYS, ILL and KCY analyzed and interpreted the data. MCH, WCH, KTL, CHW, HYS, ILL and KCY wrote, reviewed and edited the manuscript. All authors read and approved the final manuscript.