Finalized 2015 SURF Presentation - Li Ching Sheng

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)

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


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


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


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


Monolayer


Mammosphere

Notch1

(150 kDa)

β-Actin

(42 kDa)

1,25(OH)2D3


Serum-free Mammosphere Medium


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


Monolayer


Mammosphere

NFκB (64-69 kDa)

β-Actin (42 kDa)




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


Monolayer


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




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

Finalized 2015 SURF Presentation - Li Ching Sheng

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