Lentiviral shRNA screen to test the validity of a gene signature of breast cancer stem cells using high throughput mammosphere assays

Jenny Chang Labby Bhuvanesh Dave PhD

Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004

Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004

Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004

A Model For “Cancer Stem Cells” In Treatment Resistance and Disease

Recurrence

CD44+/CD24-

Observed Predictive

CD

44+/

CD

24-

0

5

10

15

20

25

30

Initial Week 3 Week 12

n=31

A Mammosphere Formation Efficiency

Observed Predictive

No.

of M

S/10

,000

cel

ls

0

10

20

30

40

50

60

70

80

90

100

110

120

130

Initial Week 3 Week 12

n=31

B

Chemotherapy Increases the Proportion of CD44+/CD24low/- Cells and

Mammosphere-Initiating Cells

Li et al (2008) JNCI 100:672-9

The Proportion of CD44+/CD24- Cells Correlates With Mammosphere Initiation

Potential

Mammospheres Are Enriched For Mammosphere-initiating Cells Compared

To The Tumor of Origin

Gene Expression Signature of Chemoresistant Mammosphere-initiating Cells

The “Signature” Is Preferentially Manifest in Claudin-Low Tumors

Design of GIPZ shRNAmir Library

Choosing a Cell Line

• In order to confirm our patient derived gene signature we looked for Claudin Low like cell lines

• Based on Joe gray’s dataset we narrowed it down to MDA-MB231, SUM-159PT & HS578T

• We chose SUM159PT because it possessed the CD44/24 population, which had been shown to have increased tumorigenic potential.

Lentiviral Screen using open bioystems library and our gene signature

We started with 493 genes and 1120 shRNA’s for this Lentiviral screen.

Screening for CSC drug targets using shRNA for gene signatureScreening for CSC drug targets using shRNA for gene signature

Library

Cherry Picking of clones

Purification of DNA

Transfection Infection

Day 1 transfect

Day 3 collect

Day 4 collect

Mammospheres

Visualize and count

Trypsinize mammospheres

HBSS + serumSpin and Resuspend in MEGM+

Visualize and count

PKH26 dye on Day4

Phase IPhase I

Phase IIPhase II

20x 20x 10x

96 Well 24 Well 6 Well

SUM159PT cells-Mammospheres plated 081408 and photographed 082008

Testing Controls

• Testing of Controls to Determine the Set of Controls to be used in the Screen

- Infectivity

- Impact on Mammosphere formation.

Experiments• Conduct the screen using SUM159PT cells with the aforementioned

controls and genes derived from our signature

• Add some of the Hedgehog and Notch sigaling pathway regulators to that gene list in order to test for things that have been the usual suspects in the cancer stem field.

• We added 40 genes from the ALDH1 derived signature from Dr. Max Wicha’s Group.

• Confirm reduction of gene expression of the control genes by real time PCR which will be eventually followed by western blots.

Results

• We conducted this screen in 14x96 well plates each time (8 times)

• We collected information on MS formation information for primary and secondary MS using Gelcount from Oxford Optronix

• We have currently analyzed the primary MS data and we got 151 unique shRNAs where MS number changes significantly p=0.05

Results of Primary MS formation screen depicted as

a Z-score of all the genes tested in the screen Z

Sco

re

Candidate Regulators p<0.05

128 shRNA’s representing 108 unique genes showed significant alteration in the MS formation Efficiency in SUM159

Increasing MSFEDecreasing MSFE

BT549 p<0.05

ex

p1

ex

p2

ex

p3

ex

p4

ex

p5

ex

p6

ex

p7

AA_Bmi1_pos2_1AA_Bmi1_pos2_8AA_GSI_pos1_11AA_GSI_pos1_8AA_GSI_pos2_10AA_GSI_pos2_8AB_BLANK1_1AB_BLANK1_3AB_BLANK1_6AB_BLANK2_12AB_BLANK2_3AB_BLANK2_6AB_BLANK2_8AB_EV1_6AB_EV1_8AB_EV2_12AB_EV2_8AC_EG5_neg1_12AC_EG5_neg1_5AC_EG5_neg1_7AC_EG5_neg1_8AC_EG5_neg2_1AC_EG5_neg2_2AC_EG5_neg2_3AC_EG5_neg2_5AC_GAPDH_neg1_1AC_GAPDH_neg1_11AC_GAPDH_neg1_4AC_GAPDH_neg1_8AC_GAPDH_neg2_13AC_GAPDH_neg2_14AC_GAPDH_neg2_2AC_GAPDH_neg2_3AC_GAPDH_neg2_4AKT1_172_0704_BDave9_H10_9AMOTL2_172_0177_BDave1_A8_1AMOTL2_172_0444_BDave1_B1_1AOX1_172_0053_BDave14_E2_14ARHGAP29_172_0427_BDave1_A1_1ARHGAP8_172_0018_BDave1_E5_1ARHGAP8_172_0305_BDave1_D2_1ARID1B_172_0194_BDave10_E6_10ATP2B1_172_0072_BDave10_G10_10ATP2B1_172_0700_BDave10_F1_10B3GALNT1_172_0471_BDave1_H5_1BCMP11_172_0363_BDave1_C6_1BIRC5_172_0086_BDave1_G2_1BIRC5_172_0255_BDave1_E4_1BIRC5_172_0288_BDave1_H6_1BIRC5_172_0474_BDave1_C11_1BMPER_172_0710_BDave14_C11_14BTG1_172_0687_BDave10_F6_10C10orf57_172_0603_BDave1_C12_1C14orf147_172_0226_BDave1_F1_1C14orf65_172_0671_BDave1_H7_1C16orf34_172_0256_BDave1_H12_1C20orf175_172_0003_BDave14_B9_14C20orf23_172_0534_BDave1_A6_1C20orf23_172_0583_BDave1_H6_1C7orf24_172_0029_BDave1_H5_1C9orf58_172_0644_BDave1_A2_1CACNA1G_172_0632_BDave10_D1_10CAPN13_172_0546_BDave1_C10_1CCDC34_172_0015_BDave2_C8_2CCDC34_172_0632_BDave2_G2_2CCDC92_172_0688_BDave10_E9_10CCL14_172_0240_BDave2_H11_2CDS1_172_0318_BDave2_C2_2CENPA_172_0315_BDave2_H6_2CENPF_172_0042_BDave2_C5_2CENPF_172_0136_BDave2_C9_2CENPF_172_0372_BDave2_F6_2CFB_172_0382_BDave2_H3_2CHMP4C_172_0310_BDave2_B10_2CHST11_172_0115_BDave10_D2_10CHST11_172_0385_BDave10_B11_10CISH_172_0066_BDave2_H6_2CISH_172_0295_BDave2_D6_2CISH_172_0688_BDave2_H10_2CNKSR3,MAGI1_172_0643_BDave2_B9_2COX6C_172_0619_BDave2_C10_2CRISP3_172_0608_BDave2_A2_2CTBP2_172_0654_BDave2_B10_2CYGB_172_0160_BDave10_A3_10DSC2_172_0406_BDave2_C6_2EFNA1_172_0354_BDave3_H9_3EIF1_172_0556_BDave10_H7_10EIF1_172_0602_BDave10_C9_10EIF4ENIF1_172_0317_BDave10_B7_10EIF4ENIF1_172_0585_BDave10_F5_10ELAVL2_172_0200_BDave3_A5_3ELAVL2_172_0472_BDave3_E8_3ELF3_172_0687_BDave3_G8_3ENAH_172_0660_BDave3_A3_3EPB41L4B_172_0178_BDave3_A10_3EPB41L4B_172_0607_BDave3_D12_3EPB41L4B_172_0712_BDave3_H12_3EPHB3_172_0704_BDave3_B6_3EPN3_172_0412_BDave3_B7_3EPPK1_172_0172_BDave3_A8_3EVA1_172_0221_BDave3_F10_3EVA1_172_0643_BDave3_B1_3EXOD1_172_0338_BDave3_F5_3F11R_172_0135_BDave3_C5_3F11R_172_0333_BDave3_F8_3F3_172_0263_BDave14_D3_14F3_172_0529_BDave14_E11_14FBXO3_172_0411_BDave3_A3_3FNBP1L_172_0608_BDave3_C9_3FOXA1_172_0217_BDave3_F1_3FOXA1_172_0506_BDave4_D1_4FRAS1_172_0621_BDave11_C3_11FRK_172_0402_BDave4_E6_4FRMD3_172_0238_BDave4_F10_4FSTL4_172_0343_BDave11_G12_11GCNT2_172_0439_BDave4_G4_4GCNT2_172_0596_BDave4_E12_4GGCX_172_0362_BDave11_G3_11GLI1_172_0145_BDave9_D10_9GLI1_172_0560_BDave9_E4_9GLI2_172_0411_BDave9_G10_9GLI3_172_0042_BDave9_H11_9GNAZ_172_0024_BDave9_E2_9GNAi2_172_0379_BDave9_G4_9GNAiO_172_0740_BDave9_F10_9GPR137C_172_0577_BDave4_G7_4GPR37_172_0484_BDave4_B1_4GRHL1_172_0013_BDave4_H9_4GRHL1_172_0539_BDave4_F7_4GRHL2_172_0074_BDave4_A11_4GRHL2_172_0625_BDave4_H2_4GSTO2_172_0118_BDave4_B11_4HES1_172_0616_BDave4_C5_4HN1_172_0354_BDave4_C4_4IFNAR1_172_0072_BDave11_H3_11IFNAR1_172_0524_BDave11_G12_11IL20RA_172_0274_BDave4_C4_4IL20RA_172_0555_BDave4_A9_4INPP4B_172_0017_BDave4_G6_4IPP_172_0312_BDave4_F7_4IRX5_172_0495_BDave4_H6_4IRX5_172_0541_BDave4_A3_4JARID1B_172_0733_BDave5_G12_5KCNK1_172_0109_BDave5_C5_5KCNK1_172_0691_BDave5_A6_5KCTD3_172_0454_BDave5_A9_5KIAA0103_172_0643_BDave5_A12_5KIAA0194_172_0473_BDave11_C8_11KIAA0194_172_0613_BDave11_E4_11KIF4A_172_0327_BDave5_D1_5KIF4A_172_0400_BDave5_F9_5KIF4A_172_0568_BDave5_D8_5KIF9_172_0589_BDave5_D4_5KLF12_172_0531_BDave11_C3_11KRTCAP3_172_0058_BDave5_F2_5LCK_172_0704_BDave14_F7_14LGR4_172_0677_BDave5_B7_5LNX2_172_0247_BDave5_G3_5LRP11_172_0617_BDave5_A8_5LRRC1_172_0165_BDave5_H12_5LUZP1_172_0183_BDave11_D6_11MAML2_172_0196_BDave11_E2_11MAP7_172_0417_BDave5_F4_5MARVELD2_172_0055_BDave5_F9_5MB_172_0159_BDave5_E5_5MGC2780_172_0438_BDave6_B12_6MST150_172_0077_BDave12_B3_12MTAC2D1_172_0320_BDave6_D6_6MTL5_172_0003_BDave6_A1_6MYCBP2_172_0234_BDave12_E6_12MYCBP2_172_0725_BDave12_H12_12MYO6_172_0374_BDave6_H7_6NEDD4L_172_0408_BDave6_G11_6NEDD4L_172_0414_BDave6_D10_6NEDD4L_172_0562_BDave6_H5_6NEK2_172_0584_BDave6_G2_6NFATC3_172_0149_BDave6_G9_6NFATC3_172_0333_BDave6_F9_6NKX3-1_172_0353_BDave6_B9_6NPNT_172_0284_BDave6_C4_6NR2F1_172_0628_BDave14_G9_14NR2F1_172_0699_BDave14_C8_14NR2F1_172_0728_BDave14_D5_14NTN4_172_0052_BDave6_E6_6NTN4_172_0252_BDave6_E4_6NTN4_172_0664_BDave6_F8_6NUP188_172_0229_BDave12_B1_12NUP188_172_0508_BDave12_F8_12OVOL2_172_0633_BDave6_H9_6PAK3_172_0499_BDave6_E10_6PAWR_172_0575_BDave6_F9_6PAWR_172_0697_BDave6_G5_6PDE4A_172_0093_BDave12_A11_12PDE4DIP_172_0634_BDave12_A10_12PDE4DIP_172_0687_BDave12_F11_12PER1_172_0125_BDave12_H10_12PGM2_172_0148_BDave7_A8_7PLCH1_172_0207_BDave7_F2_7PLS1_172_0367_BDave7_C12_7PODXL_172_0021_BDave7_C1_7PODXL_172_0649_BDave7_E2_7POLR2A_172_0059_BDave12_E10_12PPAPDC2_172_0112_BDave7_B12_7PPAPDC2_172_0114_BDave7_G6_7PPFIA3_172_0591_BDave7_C12_7PPP3CA_172_0348_BDave12_G6_12PRRG4_172_0439_BDave7_G12_7PTEN_172_0379_BDave9_H9_9PTPRCAP_172_0207_BDave14_H11_14PTTG1_172_0641_BDave7_H1_7RAB25_172_0475_BDave7_D1_7RASSF7_172_0214_BDave7_G1_7RHPN2_172_0329_BDave7_C9_7ROGDI_172_0024_BDave7_G9_7RPL39L_172_0645_BDave7_D2_7SGEF_172_0349_BDave7_H11_7SGEF_172_0550_BDave7_A9_7SH3RF1_172_0232_BDave8_G5_8SMAD1_172_0160_BDave8_H10_8SMAD1_172_0191_BDave8_C12_8SPDEF_172_0258_BDave8_H5_8SSBP2_172_0009_BDave13_B8_13SSX2IP_172_0199_BDave8_F4_8SSX2IP_172_0357_BDave8_A8_8STAT3_172_0098_BDave10_H4_10STC2_172_0337_BDave8_H9_8TACSTD1_172_0053_BDave8_D11_8TATDN1_172_0569_BDave8_B1_8TATDN1_172_0685_BDave8_A1_8TCEA3_172_0355_BDave8_D4_8TCHP_172_0191_BDave13_G10_13TCHP_172_0210_BDave13_A8_13THAP10_172_0654_BDave8_B6_8TM4SF18_172_0295_BDave8_A5_8TMED5_172_0196_BDave13_H2_13TMEM30B_172_0278_BDave8_B4_8TMSL8_172_0482_BDave8_B4_8TNRC9_172_0014_BDave8_A9_8TPD52_172_0133_BDave8_D10_8TRBV19_172_0012_BDave14_B2_14TRIO_172_0525_BDave13_E4_13TULP4_172_0215_BDave9_F9_9ULK2_172_0064_BDave9_C8_9ULK2_172_0403_BDave9_B5_9ULK2_172_0549_BDave9_F4_9VAV3_172_0261_BDave9_C1_9VIM_172_0060_BDave13_D3_13WDR40A_172_0360_BDave9_C4_9WDR40A_172_0724_BDave9_H4_9WWP1_172_0372_BDave9_B5_9WWP1_172_0492_BDave9_D5_9ZBTB16_172_0522_BDave13_G1_13ZCCHC6_172_0265_BDave13_H3_13ZCCHC6_172_0495_BDave13_F8_13ZMYM2_172_0587_BDave13_B3_13ZNF750_172_0456_BDave9_F2_9ZNF77_172_0505_BDave9_E11_9ZNF93_172_0317_BDave9_A12_9

Candidate Regulators p<0.05

BT549

Increasing MSFEDecreasing MSFE

BT549

Future Directions

• Do real time PCR of all the targets.

• Liposomal delivery of shRNA in cells/animals using nanoparticles

• Target two tumor lines based on five target gene identified from the combination of these two cell lines.