The Effects of Vitamin D on Breast Cancer Stem Cells 7-30-2015 Li Ching Sheng Mentor: Joseph Wahler Dr. Nanjoo Suh Susan Lehman Cullman Laboratory for Cancer Research
The Effects of Vitamin D on
Breast Cancer Stem Cells
7-30-2015
Li Ching Sheng
Mentor: Joseph Wahler
Dr. Nanjoo Suh
Susan Lehman Cullman Laboratory for Cancer Research
Breast Cancer
SubtypesThese tumors
tend to be…
Approximate
Prevalence
Luminal A
Estrogen receptor + and/or
Progesterone receptor +
HER2 receptor -
40%
Luminal B
Estrogen receptor + and/or
Progesteron receptor +
HER2 receptor +/-
20%
Triple negative / basal-like
Estrogen receptor -
Progesterone receptor -
HER2 receptor -
15-20%
HER2 type
Estrogen receptor –
Progesterone receptor –
HER2 receptor +
10-15%
Carey LA, Perou CM, Livasy CA, et al. Race, breast cancer subtypes, and survival in the Carolina Breast cancer Study. JAMA. 295(21):2492-2502, 2006.
Voduc KD, Cheang MC, Tyldesley S, Gelmon K, Nielsen TO, Kennecke H. Breast cancer subtypes and the risk of local and regional relapse. J Clin Oncol. 28(10):1684-91, 2010.
Carey LA. Molecular intrinsic subtypes of breast cancer. In: UpToDate. Hayes DF, Dizon DS (eds.). Waltham, MA: UpToDate, 2013.
Cancer Stem Cell Hypothesis
Figure obtained with permission from Jeffrey Yang
Recurrence!
Regression!
Stem-Like Cancer Cell Differentiated Tumor Cell
Targeted
Therapy
Conventional
Chemotherapy
Conventional
Chemotherapy
Drug that kills
tumor stem cell…
…tumor loses ability to
regenerate new cells…
Drug that kills
tumor cells…
…but not cancer
stem cell…
Vitamin D
Functions
Valdivielso JM, Cannata-
Andía J, Coll B, Fernández
E. A new role for vitamin
D receptor activation in
chronic kidney disease
American Journal of
Physiology 2009; 297, 6:
F1502-F1509
Vitamin D3
1,25(OH)2D3
Vitamin D Pathway
Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman B. The role of vitamin D in reducing cancer risk and progression Nature Reviews Cancer
14, 342–357 (2014)
Vitamin D Pathway
Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman B. The role of vitamin D in reducing cancer risk and progression Nature Reviews Cancer
14, 342–357 (2014)
Vitamin D Pathway
Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman B. The role of vitamin D in reducing cancer risk and progression Nature Reviews Cancer
14, 342–357 (2014)
Previous Studies
● Vitamin D compounds inhibit the transition of ductal carcinoma in
situ (DCIS) to invasive ductal carcinoma1
● Breast cancer stem cell-like population of MCF10DCIS was
reduced upon treatment with vitamin D compounds2
● Mammosphere formation of MCF10DCIS treated with vitamin D
analog was significantly decreased2
● Vitamin D analog suppresses tumor-initiation subpopulation of
MCF10DCIS through HES1-mediated inhibition of Notch1
signaling3
1Wahler J., So JY, et al. Inhibition of the Trasition of Ductal Carcinoma In Situ to Invasive Ductal Carcinoma by a Gemini Vitamin D Analog. Cancer Prevention
Research 2014; 10. 1158/1940-62072Wahler J., So JY, et al. Vitamin D compounds reduce mammosphere formation and decrease expression of putative stem cell markers in breast cancer.
Elsevier Journal of Steroid Biochemistry & Molecular Biology 20143So JY, Wahler J, et al. HES1-mediated inhibition of Notch1 signaling by a Gemini vitamin D analog leads to decreased CD44+/CD24-/low tumor-initiating
subpopulation in basal-like breast cancer. Elsevier Journal of Steroid Biochemistry & Molecular Biology 2014
To test for the effects of vitamin D
in a triple-negative breast cancer
cell line
Objective
To compare the effects of vitamin
D in SUM159 monolayer and
mammosphere
Approach
Aim I: To compare the
Vitamin D receptor level
in monolayer and
mammosphere
Effect of Vitamin D on Vitamin D
Receptor Level
Control 1nM 10nM 100nM 1uM
Monolayer
Control 1nM 10nM 100nM 1uM
Mammosphere
VDR (48 kDa)
β-Actin (42 kDa)
1,25(OH)2D3
24-hour treatment 6-day treatment
Ultra-low attachment plates
1.00 1.63 1.53 1.46 1.62 1.00 1.14 0.88 0.90 0.69
Aim IIa: To assess the
proliferative ability of
SUM159 treated with
different doses of Vitamin D
Effect of Vitamin D on Proliferation
in SUM159 Monolayer
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 Hour 24 Hour 48 Hour 72 Hour
Ab
so
rba
nc
e
Control
10nM
100nM
1uM
1,25(OH)2D3
Aim IIb: To examine the
colony-forming ability of
SUM159
MammosphereParent Stem Cell
Multipotent Progenitor Cell
Lineage-Restricted Progenitor Cell
Differentiated, Lineage-Committed Cell
Differentiated, Lineage-Committed Cell
A non-adherent in vitro colony-forming assay used for the quantification of stem
cell/early progenitor activity and stem cell self-renewal…
Side View of a Well in a
Ultra-Low Attachment
6-Well Plate
Monolayer SUM159 Cell Culture
The Mammosphere Formation Assay
Figure obtained with permission from Jeffrey Yang
Control 1nM 10nM 100nM 1uM
Effect of Vitamin D on SUM159
Mammosphere Formation1,25(OH)2D3
Conditions: 4,000 cells in serum-free mammosphere medium in ultra-low attachment plates;
6-day treatment
0
0.1
0.2
0.3
0.4
0.5
0.6
Control 1nM 10nM 100nM 1uM
Mam
mo
sp
here
FO
rmin
g E
ffic
ien
cy (
%)
1,25(OH)2D3 Treatment
Mammosphere Forming Efficiency of Vitamin D-treated SUM159 Cells
* *
* p<0.0001
Aim III: To examine the
proteins involved in stem
cell signaling pathways in
SUM159 monolayer vs.
mammosphere
Effect of Vitamin D on Notch1 Level
Control 1nM 10nM 100nM 1uM
Monolayer
Control 1nM 10nM 100nM 1uM
Mammosphere
Notch1
(150 kDa)
β-Actin
(42 kDa)
1,25(OH)2D3
24-hour treatment 6-day treatment
Serum-free Mammosphere Medium
Ultra-low attachment plates
1
1.00 1.05 0.92 1.03 0.92 1.00 1.01 0.85 0.71 1.00
Effect of Vitamin D on NFκB Level
Control 1nM 10nM 100nM 1uM
Monolayer
Control 1nM 10nM 100nM 1uM
Mammosphere
NFκB (64-69 kDa)
β-Actin (42 kDa)
24-hour treatment 6-day treatment
Serum-free Mammosphere Medium
Ultra-low attachment plates
1,25(OH)2D3
1.00 1.19 1.02 0.90 1.11 1.00 0.87 0.64 0.77 0.68
Effect of Vitamin D on β-Catenin Level
Control 1nM 10nM 100nM 1uM
Monolayer
Control 1nM 10nM 100nM 1uM
Mammosphere
β-Actin (42 kDa)
β-Catenin (92 kDa)
Note: different exposure times for β-Catenin (ML: 4 min, developed two days later; MS: 10 sec, developed on the first day)
1,25(OH)2D3
24-hour treatment 6-day treatment
Serum-free Mammosphere Medium
Ultra-low attachment plates
1.00 1.44 1.87 1.29 1.67 1.00 0.89 0.68 0.68 0.63
Conclusions
1. 1,25(OH)2D3 decreases Vitamin D receptor
level in SUM159 mammosphere
2. 1,25(OH)2D3 does not alter cell growth in
SUM159 monolayer
3. 1,25(OH)2D3 decreases colony-forming
efficiency in SUM159
Future Directions
● Investigate cleaved Notch1 and phosphorylated
NFκB levels
● Investigate hedgehog signaling
● Investigate the localization of β-Catenin
● Further explore the relationship between vitamin
D, β-Catenin, and the Wnt signaling pathway
Acknowledgement
Lab members SURF Program
Dr. Nanjoo Suh
Dr. Philip Furmanski
Joseph Wahler
Dr. Soumyasari Das Gupta
Dr. Minji Bak
Dr. Michael Chen
Jeffrey Yang
Sonam Chodon
Jisell Rosario
Dr. Lauren Aleksunes
Dr. Debra Laskin
Stephanie Marco
Ragena Riley
This study is funded by Rutgers
Graduate School of Biomedical
Sciences Support