Vitamin D and Breast Cancer: Does Weight Make a Difference?

Vitamin D3 (cholecalciferol, colecalciferol) is one of type of vitamin D which is made by the
skin when exposed to sunlight; it is also found in some foods and can be taken as a dietary
supplement. It is used to treat and prevent vitamin D deficiency and associated diseases,
including rickets. Vitamin D3 may have a role obesity and cancer biology. In the body,
Vitamin D3 is metabolized to the active form 1,25-dihydroxycholecalciferol (calcitriol,
1,25-(OH)2vitamin D3).

This protocol is a randomized, controlled, and blinded clinical trial in obese and non-obese
breast cancer patients in whom the effects of vitamin D supplementation will be evaluated in
the neoadjuvant setting. Changes in biomarker expression levels in blood will be assessed
from baseline to post-treatment (post-surgery).

Per protocol (see title), the treatment groups are defined as those participants with body
mass index (BMI) ≤ 25 ("non-obese") and > 25 ("obese"). Within each treatment group, dose of
Vitamin D3 was stratified between 400 IU/day (control) and 10,000 IU/day (experimental). All
analyses were typically conducted between BMI cohorts stratified by Vitamin D3 dose.

Protocol Primary Objective: "To determine whether dietary vitamin D can reverse the negative
effects of obesity and insulin resistance as reflected by changes in breast cancer gene
expression patterns in obese and non obese subjects diagnosed with breast cancer."

Protocol Secondary Objectives: "To determine whether dietary vitamin D can reverse the
negative effects of obesity and insulin resistance as reflected by serum biomarkers of
insulin resistance and adipokine secretion in obese and non obese subjects diagnosed with
breast cancer."

The following markers will be part of this study.

- Insulin-like growth factor-binding protein 3 (IGFBP-3) is a Vitamin D target, and the
most abundant of 6 different IGFBPs. The insulin-like growth factors 1 and 2 (IGF-1 and
IGF-2) binds to IGFBP-3 with high affinity, which helps lengthen the half-life of
circulating IGFs in all tissues. Through this binding action, IGFBP-3 exerts
anti-proliferative effects by blocking the ability of IGFs to activate the IGF-1
receptor (IGF1R, which stimulates cell proliferation). IGFBP-3 levels were assessed on
the basis of mRNA levels.

- p21 [also known as p21Cip1; p21Waf1, cyclin-dependent kinase inhibitor 1 (CDKI1) or
cyclin-dependent kinase (CDK)-interacting protein 1 (CDKIP1)] is the primary mediator of
p53-dependent cell cycle arrest in response to DNA damage, and is a proliferation;
apoptosis; and invasion biomarker. p21 is primarily associated with inhibition of CDK2,
but is capable of inhibiting all cyclin/CDK complexes. p21 3 levels were assessed on the
basis of mRNA levels.

- Matrix metalloproteinase-11 (MMP-11), aka Stromelysin-3 (SL-3), is involved in the
breakdown of extracellular matrix in the disease processes of metastasis and arthritis,
as well as various normal physiological processes, such as embryonic development,
reproduction, and tissue remodeling. MMP-11 levels were assessed on the basis of mRNA
levels.

- Ki-67 ("Antigen Identified By Monoclonal Antibody Ki-67", Antigen Ki67) is the protein
produced by the gene MKI67, and is a nuclear protein that is associated with cellular
proliferation and ribosomal RNA transcription. Ki-67 is a recognized biomarker for
mitotic rate and cellular, but is absent in resting (quiescent) cells (Stage G0). In
breast cancer, Ki67 identifies a subset of patients with ER-positive breast cancer that
is highly proliferative, and who derive greater benefit from adjuvant chemotherapy.
MKI67 levels were assessed on the basis of mRNA levels.

- ESR1 is the gene encoding estrogen receptor alpha (ERα), also known as NR3A1 (nuclear
receptor subfamily 3, group A, member 1), and is a nuclear receptor that is activated by
the sex hormone estrogen. ERα plays a role in the physiological development and function
of a variety of organ systems to varying degrees, including the reproductive, central
nervous, skeletal, and cardiovascular systems. Genetic polymorphisms in ESR1 have been
associated with breast cancer. ESR1 levels were assessed on the basis of mRNA levels.

- Leptin is a stimulator of adipose stromal cell estrogen synthesis and a breast cancer
growth promoter. Serum levels of leptin correlate with body mass index (BMI, a measure
of obesity) and breast cancer risk. Leptin receptors are expressed by breast cancer
cells and leptin can promote (regulate) breast cancer cell proliferation. Leptin levels
were measured by Western blot or immunohistochemical (IHC) methodology.

- Adiponectin inhibits breast cancer growth. Adiponectin receptors are expressed by breast
cancer cells and adiponectin can inhibit (regulate) breast cancer cell proliferation by
stimulating apoptosis in estrogen receptor (ER)+ Breast cancer cells and suppressing
estrogen-stimulated cell growth. Adiponectin levels were measured by Western blot or
immunohistochemical (IHC) methodology.

- Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR) is a standard
determination of insulin resistance based on fasting insulin and glucose levels.

- cRP (C-reactive protein) is an inflammation marker produced by the liver. High levels of
CRP in the blood are indicative of a wide variety of conditions, including cancer. cRP
levels were measured by Western blot or immunohistochemical (IHC) methodology.

Inclusion Criteria:

- Women who have undergone a core needle biopsy demonstrating an operable breast cancer
whom have not yet had any further therapy.

- No prior therapy for breast cancer.

- Age 18 years or older.

- Any menopausal status

- Planned surgical resection of breast cancer or repeat core biopsy tissue sampling
prior to initiation of neoadjuvant systemic chemotherapy.

- Availability of tissue blocks from initial core needle biopsy.

- Signed informed consent.

- Willing to discontinue use of all supplements containing Vitamin D for the duration of
the study, and take only the Vitamin D provided by the study.

Exclusion Criteria:

- Presence of any Metastatic lesion.

- History of parathyroid disease, hypercalcemia, or kidney stones.

- History of Selective estrogen receptor modulator (SERM) or aromatase inhibitor
therapy.

- Receiving metformin.

- History of renal failure requiring dialysis or kidney transplantation.

- Women who are known to be pregnant or who are nursing. (As vitamin D does not have
toxicity to the fetus, a negative pregnancy test is not a requirement to participate
in the study.)

- Patients planned for surgical therapy of their breast cancer or initiation of systemic
chemotherapy, that would not allow for at least 7 days of vitamin D intervention

- Any condition potentially interfering with subjects ability to comply with taking
study medication.

- Any medical condition that would potentially interfere with vitamin D absorption.

- Current participation in another research study that would increase risk to subject,
in the opinion of the investigators.

- Patients currently taking more than 2000 IU of Vitamin D.