Impact of Walnut Consumption on Gene Expression in Breast Cancer: A Clinical Trial

Consumption of walnuts has slowed breast cancer growth and reduced the risk of mammary cancer in mice. The benefit against cancer was associated with altered expression of genes for cancer growth and survival. We hypothesized that walnut consumption would alter gene expression in pathologically confirmed breast cancers of women in a direction that would be expected to decrease breast cancer growth and survival, as was seen in mice.

The study was a nonplacebo, 2-arm, clinical trial. Women with breast lumps large enough for research and pathology biopsies were recruited and randomized to walnut consuming or control groups. Immediately after biopsy collection, women in the walnut group began to consume 2 oz of walnuts per day until follow-up surgery. Pathological studies confirmed that lumps were breast cancer in all women who remained in the trial.

At surgery, about 2 weeks after biopsy, additional specimens were taken from the breast cancers. Changes in gene expression in the surgical specimen compared to baseline were determined in each individual woman in walnut-consuming (n = 5) and control (n = 5) groups. RNA sequencing expression profiling revealed that expression of 456 identified genes was significantly changed in the tumor due to walnut consumption. Ingenuity Pathway Analysis showed activation of pathways that promote apoptosis and cell adhesion, and inhibition of pathways that promote cell proliferation and migration. These results support the hypothesis that, in humans, walnut consumption could suppress growth and survival of breast cancers.

Diet is thought to make a large contribution to risk for developing cancer [1,2], with some dietary components increasing and some components decreasing risk. Many research studies involving cancer prevention strategies have used isolated food components to ascertain that the component had cancer preventive properties. Although it is scientifically desirable to use individual components to be able to define the cancer prevention properties of the specific component, people routinely consume whole foods. When combined, the multiple components of whole foods could synergize or antagonize, either increasing or decreasing the effects of the individual components [3]; thus, it is important to test the effects of whole foods on cancer.

Cell culture studies can provide potential mechanisms for slowed tumor growth of individual food components. One cell culture study used extracts from walnuts, and the authors reported that components of walnuts, especially alphalinolenic acid and beta-sitosterol, are absorbed and either directly or indirectly decreased proliferation of MCF-7 cells [4]. Another cell culture study reported that a potent antioxidant, ellagic acid, present in high amounts in walnuts and many fruits, can inhibit cell proliferation and induce apoptosis [5]. Oxidative stress and the inflammation induced by oxidative stress have been proposed as key promoters of cancer [6]. The abundant and varied forms of antioxidants in walnuts [5,7-14] are likely to be critical to the cancer growth suppression and apoptosis inducing mechanisms of walnuts.

Methods and Materials

However, cell culture studies are not usually designed to test the effects of interactions of components of whole foods. Preclinical studies can provide tumor development and growth data and might better assess the potential effects of a whole food. We conducted 2 preclinical studies to determine whether consumption of walnuts might affect cancer growth and development. In study 1, we found that addition of a small amount of walnuts (the human equivalent of 2 oz/d, calculated as a fraction of total calories) to the diet of mice significantly slowed the growth of implanted MDA-MB 231 human breast cancers [15]. Because we used the whole walnut meat rather than an isolated component, we could not identify the specific component(s) of the walnut that was (were) effective at slowing cancer growth.

Components of walnuts that have individually been shown to slow cancer growth include α-linolenic acid (an 18C omega 3 fatty acid) [16], beta-sitosterol [17], vitamin E [18], and melatonin [19]. We also could not identify a specific mechanism(s) for the slower tumor growth, but proliferation was significantly decreased and apoptosis and antioxidative capacity were slightly increased.

The Marshall University Office of Research Integrity has an Institutional Review Board, which reviews and monitors all human subject research conducted at Marshall University, St Mary's Medical Center, Cabell Huntington Hospital, and the Edwards Cancer Center. The research protocol and participant informed consent were approved by the Institutional Review Board (protocol number 339384-3). This study was not listed at ClinicalTrials.gov.

Potential study participants were identified from records review by the Research Study Nurse prior to their appointment for a diagnostic biopsy. At the appointment time, the potential participant was interviewed by the study nurse, the study was explained, and informed consent was obtained. The physician obtained 1 or 2 additional biopsies for research use when the biopsy was obtained for pathology studies.

2.1.1. Inclusion criteria All subjects (1) were female and with a breast mass that, according to standard of care, was to be biopsied for diagnosis and was large enough to obtain the needed biopsies for pathology and research; (2) understood and were willing to sign the informed consent form; (3) had an Eastern Cooperative Oncology Group performance status of 0 or 1 (0: fully active, able to carry on all predisease performance without restriction; 1: restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, eg, light housework, office work); (4) were between 18 and 90 years of age; and (5) were recruited as available without regard to race or ethnicity.

2.1.2. Exclusion criteria Excluded persons were (1) those who do not like or who were allergic to walnuts or other tree nuts; (2) those with any metabolic disease that could be affected by walnut consumption; (3) those with a life expectancy less than 6 months; and (4) those who were pregnant (to prevent confounding due to pregnancy hormonal factors).

2.2. Clinical protocol Subjects were consented at their first visit and were randomized into treated (consume walnut) or control (no added walnuts) groups. Routine clinical data were recorded (age, weight, height, family history, etc). A 5-mL blood specimen in ethylenediaminetetraacetic acid was collected for the research laboratory. After the initial biopsy, the subject was asked to continue to consume the usual diet and to not change consumption of any medication or supplements. If she was randomized to the walnut group, the subject was given thirty 1-oz packages of walnuts and was asked to consume 2 packages (2 oz) of walnuts daily and to return remaining packages for counting.

If needed, due to extended time for the clinical workup, the subject was given additional packages of walnuts to allow for continued consumption of 2 oz of walnuts per day until surgery (about 2 to 3 weeks). Control group subjects were asked to not intentionally consume walnuts. At the conclusion of the study, each subject was asked to identify whether any changes were made to the usual diet especially in the areas of fruits, vegetables, nuts, or supplement consumption and fats used in cooking and whether walnuts were consumed (walnut group) or not (control group).