Supplementary Figure 1. Expression of hormone receptors in cultured T47D and MCF7 cells. A) Immunocytochemistry of estrogen receptor-α (ERα), progesterone receptor (PR), or glucocorticoid receptor (GR). Green stain – hormone receptor expression; blue stain (nuclei; DAPI). B) Immunohistochemistry of formalin-fixed, paraffin-embedded pellets of cultured T47D and MCF7 cells stained for estrogen receptor-α (ERα), progesterone receptor (PR), or glucocorticoid receptor (GR). Red stain – hormone receptor expression; green stain – cytokeratin; blue stain – DAPI (nuclei).

Supp Figure 1

GR ERPR

T47

DM

CF

7

10x

A

BGR ARMRPR

MC

F7

T47

D

A

T47D, CK5/Dapi

1µM

10x

Veh 10µM

B

Supp Figure 2

MCF7

0 0.1 10

1

2

3

4

Dexamethasone (µM)

% C

K5+

cel

ls (

FIT

C+

) ***

***

0 0.1 1 100

2

4

6

8

Dexamethasone (µM)

% C

K5+

cel

ls (

FIT

C+

)

T47D

****

***

Supplementary Figure 2. A) Induction of CK5+ cells in cultured T47D and MCF7 lines in response to increasing concentrations of Dex for 6 days. B) Representative images of immunocytochemistry for CK5 in T47D cultures exposed to 0, 1 or 10 µM Dex for 6d.

Supp Figure 3

T47D

Veh Dex Prg Ald0

2

4

6

8Veh

RU-486

% C

K5+

cel

ls (

FIT

C+

)

****

****

MCF7

% C

K5+

cel

ls (

FIT

C+

)

Veh Dex Prg Ald0

5

10

15 Veh

RU-486***

Supplementary Figure 3. Flow cytometric analysis of CK5+ cells (percent) in T47D (left) and MCF7 (right) cultures treated with Veh, Dex, Prg or Ald in the absence and presence of RU-486 for 6 days. Mean ± SEM are shown.

C

A B

Supp Figure 4

1 2 3 4 5 6 >70

20

40

60

80

100 2d

5d

***

*

Number of CK5+ cells per cluster

Nu

mb

er o

f C

K5+

clu

ster

s

0d 1d 4d 6d0

2

4

6

8

10

% C

K5+

cel

ls (

FIT

C+

)

*

***

****

Dexamethasone (days)

T47D, CK5/Dapi

2d 5d0dDex

40x

Supplementary Figure 4. Time course of glucocorticoid-induction of CK5+ cells in T47D and MCF7 cell cultures. A) Flow cytometric analysis of % CK5+ cells in T47D cultures continue to increase through 6 days. CK5+ cells expand in clusters with larger clusters appearing by Day 5 compared to Day 2 as quantified in (B) with representative immunocytochemistry images showing CK5 (red) and DAPI (blue) (C). Flow cytometric analysis of % CK5+ cells in MCF7 cultures showing rapid Dex-induction of CK5+ cells as early as 2 days (D). qRT-PCR analysis of time course of Dex induction of CK5 transcripts in T47D cultures.

D MCF7

%C

K5+

cel

ls (

FIT

C+

)

0d 2d 5d0

5

10

15

E T47D

4hr 16hr 24hr 48hr0

5

10

15

20 VehDex

**

**

CK

5 m

RN

A (

Fol

d in

duct

ion)

Supp Figure 5

Supplementary Figure 5. T47D cell tumor xenograft growth data prior to (Ctrl) and post- (Tam) tamoxifen treatment in mice. Xenografts were allowed to establish until day 40 (Ctrl tumors; red arrow), when half of the mice were sacrificed and tumors collected for CK5 staining (n=5). The remaining mice (n=5) then received slow release tamoxifen pellets (10mg/90 day release) s.c. on Day 45 through Day 105, at which time the mice were sacrificed and tamoxifen-resistant tumors (blue arrow) collected for CK5 staining.

Time (Days)

10 20 30 40 50 60 70 80 10090

Tum

or

volu

me

(mm

2)

Tamoxifen

800

600

400

200

0

Ctrltumors

Tam-resistant tumors

Supp Figure 6

Veh Dex

CK

5/B

cl6

C

BAT47D

0 10Dex (μM) 3 10.30.10.01 0.03

Bcl6

GAPDH

4 8 16 24 480

MCF7

Dex (h)

Bcl6

GAPDH

MCF7

20x

Supplementary Figure 6. A) Dose-dependent induction of BCL6 protein by dexamethasone in T47D cell cultures by western blot. B) Time course of Bcl6 protein expression in MCF7 cells treated with Dex. C) Representative image showing induction of Bcl6 (green) and CK5 (red) in MCF7 cells. D) Western blot showing efficacy of Bcl6 protein knockdown in T47D cells treated with or without dexamethasone by increasing doses of two independent shRNAs delivered by lentiviral infection. NTC – non-target control shRNA.

Dex

Bcl6

ShRNA

- + + + + +

NTC Bcl6

Bcl6-shRNA-1

Bcl6-shRNA-2

D

Veh

Dex

Prl

Dex/Prl

Time (h) 0 1 8 16 24 48 72 0 1 8 16 24 48 72

Bcl6 pStat5C

Dex

Ald

Bcl6

GAPDH

Bcl6

GAPDH

Steroid

Prl (nM) 0.4 0.9 2 4 9 20- - 20

- -+ + + + + + +

B

ZR75.1

MCF7

T47D

Dex - + +-

Prl

Bcl6 GAPDH

+ +- -

- + +-

+ +- -

ASupp Figure 7

D

Veh Prl

Bcl6

GAPDH0Ald(µM) 10.30.10.01 0.030.001 0.003 0 10.30.10.01 0.030.001 0.003

10.30.10.01 0.03

Bcl6

GAPDH

Dex(µM) 103 0 10.30.10.01 0.03 1030

MC

F7

T47

D

Supplementary Figure 7. Prolactin effectively suppresses basal and 3-ketosteroid-induced Bcl6 protein in various ERα+ human breast cancer cell lines. Western blot showing effective knockdown of basal and Dex-induced Bcl6 protein in T47D, MCF7 and ZR75.1 cultured cell lines (A). Western blot showing effective knockdown of basal Bcl6 protein or Bcl6 protein induced by Dex or Ald in T47D cells (B). Western blot showing sustained knockdown of basal Bcl6 protein or Bcl6 protein induced by Dex in T47D cells, with parallel sustained tyrosine phosphorylation of Stat5 (C). Western blot showing effective knockdown of Bcl6 protein even at high doses of Dex (upper panels) or Ald (lower panels) in MCF7 cells (D).

Supp Figure 8

0-10

310

310

410

5

α-CK5 (Alexa Fluor-488)

0-10

310

310

410

5

α-CK5 (Alexa Fluor-488)

VehDexPrgAldDHTEst

PrlDex+PrlPrg+PrlAld+PrlDHT+PrlEst+Prl

T47DA

T47D

% C

K5+

cel

ls (

FIT

C+

)

Veh Dex Prg Ald0

5

10

15 Veh

Prl

EGF

Prl+EGF

BMDA-MB-231

% C

K5+

cel

ls (

FIT

C+

)

Veh Dex Prg Ald DHT Est0

2

4

6

8

10Veh

Prl

C

Supplementary Figure 8. Representative flow cytometry data showing reversal of CK5 induction by 3-ketosteroids Dex, Prg, Ald and DHT by prolactin cotreatment in T47D cells (A). Flow cytometry data documenting effective suppression of Dex-induced CK5 cell population in T47D cells by prolactin but not EGF (B). Flow cytometry data showing that the ERα-negative mesenchymal cell line MDA-MB-231 does not respond to 3-ketosterids with CK5+ cell induction and also shows absence of suppressive prolactin response (C).

Luminal A Luminal B

Her2+ TNBC

GR/CK/DAPI

Supp Figure 9

20x

Supplementary Figure 9. Representative images of GR expression in breast cancer subtypes by immunofluorescence staining. GR (red), pan-cytokeratin (green), and DAPI (blue) (A). Relative quantitative expression of ER, PR and CK5 in ER+ breast cancer subgroups ER+/CK5-/Ki67lo (Luminal A-like), ER+/CK5-/Ki67hi (Luminal B-like), and ER+/CK5+ (Luminobasal-like) (B).

ERα+/CK5-/Ki67lo ERα+/CK5+ERα+/CK5-/Ki67hi

CK5

PRER

2.0

3.5

4.0

4.5

5.0

2.5

3.0

Rel

ativ

e Q

uan

tita

tive

Exp

ress

ion

(lo

g) ***

****

p <0.001p <0.05

***

A

B

*

Supp Figure 10

A

B

Supplementary Figure 10. Recursive partitioning regression tree models were developed for CK5 and BCL6 mRNA expression levels (A) or CK5 and BCL6 protein expression levels (B) where splitting decisions are based on optimization of the log-rank statistic, and plotted using partykit package in R.  End nodes are then amalgamated based on homogeneity of hazard ratio, here labeled as L (low) and H (high).    

6.465 6.465

9.537 9.537

8.019 8.019

8.952 8.952

7.233 7.233

7.684 ≥ 7.684

n = 9

0 4 8 120

0.20.40.60.8

1

BCL6

8.556 8.556

n = 22

00.20.40.60.8

1n = 20

00.20.40.60.8

1n = 23

00.20.40.60.8

1

9.069 9.069

9.124 9.124

n = 8

00.20.40.60.8

1

9.615 9.615

n = 10

00.20.40.60.8

1n = 32

00.20.40.60.8

1n = 13

00.20.40.60.8

1n = 8

00.20.40.60.8

1n = 13

00.20.40.60.8

1n = 12

00.20.40.60.8

1

BCL6

BCL6

BCL6

BCL6

BCL6

CK5

CK5

CK5

CK5

≥

≥

≥

≥

≥

≥

0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12

≥

≥

≥

6

8

5

4

13

12

15

3

2

1

L L L L H H H H H H H

1565.717 1565.7172

940.119 940.119

n = 13

0 100 2000

0.20.40.60.8

1

813.704 813.7045

750.706 750.706

n = 24

00.20.40.60.8

1

3719.381 3719.381

n = 9

00.20.40.60.8

1

637.166 637.16610

8755.707 8755.707

n = 10

00.20.40.60.8

1n = 14

00.20.40.60.8

1n = 74

00.20.40.60.8

1n = 17

00.20.40.60.8

1n = 9

00.20.40.60.8

1

CK5

CK5

BCL6

≥

≥

≥

≥

≥

≥

≥

0 100 200 0 100 200 0 100 200 0 100 200 0 100 200 0 100 200 0 100 200

BCL6

7

9

CK5

4

CK5

1

CK5

HHHLLLLL