Vitamin D Signals Via a Novel Pathway to Inhibit Metastasis

Institution: Stanford University
Investigator(s): Brian  Feldman , M.D., Ph.D. -
Award Cycle: 2013 (Cycle 19) Grant #: 19IB-0103 Award: $236,068
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2013)

Non-technical overview of the research topic and relevance to Breast Cancer:
Many studies, but not all, indicate that Vitamin D (VitD) deficiency increases the risk of developing breast cancer (BCa) and also that VitD deficient women who develop BCa have worse outcomes. The mechanism for the protection by VitD adequacy compared to deficiency is not clear. Yet many women are taking VitD supplements in the hopes that it will improve survival. Most studies that attempt to understand the mechanism of VitD benefit are directed at the ability of VitD to inhibit the growth of the BCa with little effort directed at reduction or prevention of metastases, the reason BCa is lethal. We have discovered that VitD robustly increases the expression of a gene called Klf17 that has been elegantly shown to protect against metastases in mice. This unique finding suggests that one element of VitD protection and improved outcome is the stimulation of Klf17, a “metastasis protector”.

The question(s) or central hypotheses of the research:
Our proposal is directed at elucidating whether VitD stimulation of Klf17 will impart protection from metastases in a mouse model where human BCa cells are placed in the breast fat pad of mice and the growth and metastatic potential of the BCa are investigated. The major questions to be addressed are: (1) to ascertain whether VitD reduces the ability of BCa to metastasize, (2) to determine whether the mechanism is due, in part, to VitD stimulation of the metastasis protector Klf17 and (3) to find out if we detect VitD stimulation of Klf17 in human BCa specimens.

The general methodology:
We have obtained or made in our lab several mouse and human BCa cells that glow because they express the enzyme luciferase (luc). This advance will allow us to follow the growth and spread of luc-labeled BCa in live mice using a special camera that detects bioluminescence. Thus we can place these cells in the mouse breast fat pad and sequentially follow the course of their growth and spread by repeated imaging of the live mice. The mice will be made VitD deficient with a special diet and compared to mice on a normal diet and to mice treated with calcitriol, the hormonally active form of VitD. This will determine whether VitD deficiency accelerates metastasis and whether calcitriol prevents, reduces or delays metastasis. A second set of mice will duplicate these conditions and will receive the same BCa cells but the Klf17 gene wil be blocked from functioning. This experiment will demonstrate whether the mechanism for reduced metastasis is indeed due to VitD stimulation of Klf17 or to other pathways. In a separate aim, the grant will study the ability of VitD to stimulate Klf17 expression in human BCa specimens from women treated with high doses of VitD or placebo in the interval between their diagnostic biopsy and surgical excision of their BCa. This will be carried out in a cost-effective way using specimens we have accumulated from another trial.

Innovative elements of the project:
In order to accomplish our goal, we have developed several innovative state-of-the-art approaches: (1) We have identified Klf17, a major suppressor of BCa metastasis, as a target gene of calcitriol and dietary VitD. (2) We have made or obtained luc-labeled mouse and human BCa cell lines, the growth of which can be followed by a sensitive in vivo bioluminescent imaging technique. This will dramatically improve our ability to detect and quantify metastatic disease. (3) We will use these luc-labeled BCa cells to study acceleration of metastasis by vitD deficiency and suppression of metastases by calcitriol therapy and determine whether the beneficial actions depend on the regulation of Klf17 expression in the BCa. (4) We are currently establishing a unique tumor bank of BCa specimens from an ongoing CBCRP-funded clinical trial in BCa patients receiving high dose VitD or placebo in the interval between biopsy and surgery. The trial will provide human BCa tissue specimens for analysis of VitD-induced changes in the Klf17 expression.

Using these innovative tools we will test our hypothesis that VitD directly stimulates Klf17 and thereby inhibits BCa metastasis. Establishing this connection would pioneer both a new paradigm in our understanding of the regulation of BCa metastasis as well as provide a foundation to pursue novel therapeutic approaches targeting this pathway to improve the prognosis of BCa patients. We make the innovative speculation that regulation of Klf17 by VitD provides a mechanistic explanation for the link between VitD deficiency and poor prognosis in BCa patients. Further, that levels of Klf17 expression in tumors may be a useful biomarker of BCa prognosis. These data will provide the basis for proposing a large intervention trial adding VitD to current therapies to determine whether the combination therapy improves BCa outcomes.

Progress Report 1 (2014)

Our proposal investigates the ability of calcitriol, the active hormonal form of vitamin D, to reduce breast cancer metastasis. Our hypothesis is based on a recent discovery that the Klf17 gene is a metastasis inhibitor in breast cancer models and our findings that calcitriol induces the expression of Klf17 in cultured breast cancer cells. The grant has two Specific Aims. Specific Aim I proposes to investigate the ability of calcitriol to inhibit breast cancer metastasis in a mouse model and assess the role of Klf17 regulation by calcitriol in its anti-metastatic actions. Specific Aim II plans to analyze the expression of Klf17 pathway genes in human breast cancer tissue specimens from BCa patients treated with high dose Vitamin D in an ongoing trial funded by a different CBCRP grant.

In Specific Aim I we used the highly metastatic 4T1 mouse breast cancer cell line in a mouse model. Luciferase (luc)-labeled 4T1 cells were implanted in the breast fat pads of BALB/c female mice. We monitored tumor growth and metastases development using sensitive bioluminescent imaging (BLI) of mice following luciferin injections. We utilized two experimental models. In Study #1 the mice were treated with substantial doses of calcitriol or vehicle and metastases were monitored by BLI. In Study #2 the mice were pretreated with calcitriol or vehicle for 4 weeks prior to tumor implantation and treatments were continued for 4 weeks post-implantation. In both models we noted a modest suppressive effect of calcitriol to reduce the rate of growth of the primary tumors. We did not find a significant reduction in metastases. However, we did find that calcitriol elicited a favorable gene expression profile in the primary tumors reducing genes thought to be pro-metastatic such as Id-1, a down-stream target of Klf17. Calcitriol also inhibited other proinflammatory genes and stimulated proliferation-inhibitor genes. Because the 4T1 cell line is so aggressive, we wondered whether inhibition of its ability to metastasize was too high a bar and we decided to study a similar but less aggressive breast cancer cell line, 168FARN, derived from the same spontaneously arising mouse breast tumor. These cells spread locally from the primary site in the breast fat pad to lymph nodes but do not metastasize widely. Our preliminary data show that calcitriol causes a significant inhibition of the growth of these cells as well as substantially induces Klf17 expression while decreasing that of Id-1 in these cells. Our strategy is to knock down VDR expression in 168FARN cells to develop clones of these cells lacking VDR expression and determine whether the loss of calcitriol signaling will increase the ability of these cells to metastasize to distal sites when implanted into the mammary fat pads of mice. These studies have now been initiated.

In Specific Aim II, the clinical trial is ongoing in women diagnosed with breast cancer, comparing those treated with 10,000 IU of vitamin D to those receiving 400 IU of vitamin D (placebo) in the neo-adjuvant period between biopsy and surgery. Specimens from the biopsy and surgery will be examined to determine whether Klf17, Id-1 or other genes involved in metastasis are appropriately regulated in human breast cancer by treatment with vitamin D. The bioinformatics analysis will begin when the final recruitment is completed.

Tumor Autonomous Effects of Vitamin D Deficiency Promote Breast Cancer Metastasis

Vitamin D mitigates the adverse effects of obesity on breast cancer in mice. doi: 10.1530/ERC-15-0557. Epub 2016 Jan 27
Periodical:Endocrine-Related Cancer
Index Medicus: Endocr Relat Cancer
Authors: Swami S, Krishnan AV, Williams J, Aggarwal A, Albertelli MA, Horst RL, Feldman BJ, Feldman
Yr: 2016 Vol: 23 Nbr: 4 Abs: Pg:251-64

Tumor Autonomous Effects of Vitamin D Deficiency Promote Breast Cancer Metastasis.
Index Medicus: Endocrinology
Authors: Williams JD, Aggarwal A, Swami S, Krishnan AV, Ji L, Albertelli MA, Feldman BJ
Yr: 2016 Vol: 157 Nbr: 4 Abs: Pg:1341-7

Inhibition of Mouse Breast Tumor-Initiating Cells by Calcitriol and Dietary Vitamin D. doi: 10.1158/1535-7163.MCT-15-0066. Epub 2015 May 1
Periodical:Molecular Cancer Therapeutics
Index Medicus: Mol Cancer Ther
Authors: Jeong Y, Swami S, Krishnan AV, et al.
Yr: 2015 Vol: 14 Nbr: 8 Abs: Pg:1951-61