ACTIVATION OF BP1 IS ACTIVATION OF BP1 IS ASSOCIATED WITH ASSOCIATED WITH

AGGRESSIVE BREAST AGGRESSIVE BREAST CANCERCANCER

PATRICIA BERG, PH.D.

pBP1 IS AN ISOFORM OF THE pBP1 IS AN ISOFORM OF THE DLX4 GENEDLX4 GENE

CHASE ET AL, 2002. MOL. CELL. BIOL. 22:2505-2514

NOTE: BP1 AND DLX4 ARE NOT INTERCHANGEABLE NOTE: BP1 AND DLX4 ARE NOT INTERCHANGEABLE TERMSTERMS

BP1BP1

DLX7DLX7

DLX4DLX4

CHARACTERISTICS OF BP1CHARACTERISTICS OF BP1

BELONGS TO THE DISTAL-LESS (DLX) FAMILY

DLX GENES ARE HIGHLY CONSERVED: FOUND IN DROSOPHILA, ZEBRAFISH, FROGS, CHICKENS, MICE AND HUMANS

IMPORTANT FOR CRANIOFACIAL, LIMB, TOOTH AND OCULAR DEVELOPMENT IN MICE

CHARACTERISTICS OF BP1CHARACTERISTICS OF BP1(CONT)(CONT)

ENCODES A TRANSCRIPTION FACTOR THAT REGULATES CASCADES OF GENES, INCLUDING ONCOGENES

A POTENTIAL ONCOGENE ACTIVATED IN A NUMBER OF DIFFERENT TYPES OF CANCER

ACTIVATION OF BP1 IN CANCERACTIVATION OF BP1 IN CANCER

BREAST CANCER – 80% (FU ET AL, 2003)

PROSTATE CANCER – 70% (SCHWARTZ ET AL, 2005)

ACUTE MYELOID LEUKEMIA – 63% (HAGA ET AL, 2000)

ACTIVATION OF BP1 IN CANCER ACTIVATION OF BP1 IN CANCER (CONT)(CONT)

OVARIAN CANCER – ~ 65% (and HARA ET AL, 2007)

LUNG CANCER – ~ 45% (and YU ET AL, 2008)

COLON CANCER – ~40%

Berg, PE and Kirolikar, S. 2011. Atlas Genet Cytogenet Oncol Haematol. 15: 658-661.

GENES INVOLVED IN NON-GENES INVOLVED IN NON-HEREDITARY BREAST CANCERHEREDITARY BREAST CANCER

GENE FREQUENCY

HER-2 20 – 30%

C-MYC 20 – 30%

CYCLIN E 30%

EGFR 25-35%

CYCLIN D1

BP1

50% (20% AMPLIFIED)

80%

ASSOCIATION BETWEEN BP1 mRNA EXPRESSION AND

CLINICO-PATHOLOGICAL DATA

BP1 mRNA Factors Negative Positive p value

ER Status 0.03Negative 0 (0%) 18 (100%)Positive 7 (27%) 19 (73%)

PR Status 0.02Negative 1 (4.0%) 24 (96%)Positive 6 (33%) 12 (67%)

Race 0.04Caucasian 6 (43%) 8 (57%)African American 3 (11%) 25 (89%)

FU et al, 2003. Br. Ca. Res. 5: 82-87.

BP1 AND BREAST CANCER BP1 AND BREAST CANCER PROGRESSIONPROGRESSION

MAN ET AL, 2005. BR. CA. RES. & TREAT. 90: 241-247.MAN ET AL, 2005. BR. CA. RES. & TREAT. 90: 241-247.

BP1 and ProgressionBP1 and ProgressionNORMAL

HYPERPLASIA (IDH)

DUCTAL CARCINOMA IN SITU (DCIS)

INVASIVE DUCTAL CARCINOMA (IDC)

0%

21%n=70

46% n=100

81% n=100

N

0% n=30

SUMMARYSUMMARY

AS TUMOR PROGRESSION OCCURS, THERE IS AN INCREASE IN

1. THE PERCENTAGE OF BP1 POSITIVE TUMORS

2. THE PERCENTAGE OF BP1 POSITIVE CELLS PER TUMOR

3. BP1 STAINING INTENSITY (BP1 EXPRESSION)

INFLAMMATORY BREAST INFLAMMATORY BREAST CANCERCANCER

RAPIDLY PROGRESSIVE HIGHLY ANGIOGENIC AND INVASIVE 5 YR SURVIVAL LESS THAN 45% VS 86% IN

NON-IBC LESS THAN HALF HAVE A DISCRETE MASS REPRESENT 1-5% OF ALL BREAST CANCERS

IN CAUCASIANS AND 10% IN AFRICAN AMERICANS

BP1 AND INFLAMMATORY BREAST BP1 AND INFLAMMATORY BREAST CANCERCANCER

100% OF IBC TUMORS WERE BP1 POSITIVE (N=45) WITH 95% OF CELLS POSITIVE

9 PAIRED METASTATIC LYMPH NODES AVAILABLE; ALL WERE BP1 POSITIVE

TUMOR CELLS IN BLOOD VESSELS AND LYMPHATIC CHANNELS WERE BP1 POSITIVE

Man et al, 2009. Ca. Biomarkers 5: 9-17. Man et al, 2009. Ca. Biomarkers 5: 9-17.

BP1 EXPRESSION IN LYMPHATIC BP1 EXPRESSION IN LYMPHATIC CHANNELS AND BLOOD VESSELSCHANNELS AND BLOOD VESSELS

LYMPHATIC CHANNELSLYMPHATIC CHANNELS BLOOD VESSELSBLOOD VESSELS

HOW IS BP1 ACTIVATED?HOW IS BP1 ACTIVATED?

A. A. DNA AMPLIFICATIONDNA AMPLIFICATION

B. ESTRADIOLB. ESTRADIOL

A. DNA AMPLIFICATIONA. DNA AMPLIFICATION

AMPLIFICATION OF BP1 WAS OBSERVED BY FISH IN 22% OF PRIMARY BREAST TUMORS AND IN 24% of MATCHED SLN METASTASES FROM PATIENTS WITH INVASIVE DUCTAL BREAST CANCER*

NOTE: BP1 MAPS TO 17Q21-22**

* * TORRESON ET AL, 2014; CAVALLI ET AL, 2008.TORRESON ET AL, 2014; CAVALLI ET AL, 2008.

** FU ET AL, 2001. GENE 278: 131-139. ** FU ET AL, 2001. GENE 278: 131-139.

FISH analysis of FFPE sections of a primary breast tumor (left) FISH analysis of FFPE sections of a primary breast tumor (left) and SLN metastasis (right) from one patient showing and SLN metastasis (right) from one patient showing

amplification of the amplification of the BP1BP1 gene gene

FISH ANALYSIS OF BP1FISH ANALYSIS OF BP1

CORRELATION BETWEEN BP1 CORRELATION BETWEEN BP1 AMPLIFICATION AND AMPLIFICATION AND

EXPRESSIONEXPRESSION

ALL CASES SHOWING BP1 AMPLIFICATION ALSO SHOWED BP1 EXPRESSION BY IHC

BP1 POSITIVITY WAS ALSO SEEN IN CASES WITHOUT AMPLIFICATION

BP1 AND HER2BP1 AND HER2

BP1 AND HER2 MAP NEAR EACH OTHER

AND ARE CO-AMPLIFIED 78% OF THE TIME IN PRIMARY TUMORS AND 100% OF THE

TIME IN SLN

B. ESTRADIOL STIMULATES BP1 IN MCF7 CELLS

ERα BINDS TO BP1 INTRON I

ChIPChIP

1. LADDER

2.3 INPUT CONTROL FOR ERE ELEMENT IN EXON I AND INTRON I

4.5 AFTER IP WITH ERα AB

BP1 ACTIVATES ONCOGENESBP1 ACTIVATES ONCOGENESGENE ASSOCIATED WITH

MMP9 INITIATION, INVASION, METASTASIS

BCL-2 ANTI-APOPTOTIC (Stevenson et al, 2007)

C-MYC PROLIFERATION, DIFFERENTIATION, APOPTOSIS

(Trinh et al, 2011)

MET

VEGF

GROWTH, ANGIOGENESIS, METASTASIS

ANGIOGENESIS (Hara et al, 2007)

BP1 EXPRESSION IS BP1 EXPRESSION IS ASSOCIATED WITH ASSOCIATED WITH AGGRESSIVENESS AGGRESSIVENESS

MEASURES OF AGGRESSIVENESSMEASURES OF AGGRESSIVENESS

BP1 INCREASES:BP1 INCREASES:

GROWTH IN THE ABSENCE OF SERUM

GROWTH IN SOFT AGAR (ANCHORAGE INDEPENDENT GROWTH)

INVASION THROUGH MATRIGEL

CHANGES IN TUMOR FORMATION IN NUDE MICE

O4

O2

V1

D4 D13

GROWTH IN SOFT AGARGROWTH IN SOFT AGAR

CELL LINES OVEREXPRESSING BP1 WERE CELL LINES OVEREXPRESSING BP1 WERE INJECTED INTO THE FAT PADS OF NUDE INJECTED INTO THE FAT PADS OF NUDE

MICEMICE

Submitted Submitted

EFFECT OF BP1 ON TUMOR EFFECT OF BP1 ON TUMOR GROWTH IN MICEGROWTH IN MICE

ESTROGEN INDEPENDENT ESTROGEN INDEPENDENT TUMOR FORMATION IN MICETUMOR FORMATION IN MICE

Cell Type Injected Estrogen added

Absence of Estrogen

MCF-7/EV-1 4/10 (40%) 0/10 (0%)

MCF-7/BP1-2 10/15 (67%) 2/10 (20%)

MCF-7/BP1-4 7/11 (64%) 2/10 (20%)

BP1 UPREGULATES ERBP1 UPREGULATES ERαα BOTH BOTH DIRECTLY AND INDIRECTLYDIRECTLY AND INDIRECTLY

SUBMITTEDSUBMITTED

DIRECT: BP1 ACTIVATES ERDIRECT: BP1 ACTIVATES ERαα RNA RNA BY BINDING TO IVS1BY BINDING TO IVS1

INDIRECT: BP1 STABILIZES ERINDIRECT: BP1 STABILIZES ERαα BY BY UPREGULATING p300 UPREGULATING p300

(p300 ACETYLATES ER(p300 ACETYLATES ERαα))

p300

ACTIN

O1V1

mRNAmRNA

PROTEINPROTEIN

INDIRECT: BP1 AND BRCA1INDIRECT: BP1 AND BRCA1

BRCA1 INHIBITS ERα PROTEIN VIA UBIQUITINATION

BP1 REPRESSES BRCA1 BY BINDING TO ITS FIRST INTRON (Kluk et al, 2010), WHICH IS PREDICTED TO FURTHER INCREASE THE LEVEL OF ERα

MODELMODEL

CONCLUSIONSCONCLUSIONS

BP1 EXPRESSION MAY BE PROGNOSTIC IN BREAST CANCER

BP1 IS A NEW POTENTIAL THERAPEUTIC TARGET

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSGWUMCSAURABH KIROLIKAR JHUSANKET AWATE JUDITH KARPDANIEL RHEEY FARHAD VESUNABIN JIN HUANG VENU RAMANKELLIE YAMANEYASSI FALLAH AFIP YAN-GAO MANARNOLD SCHWARTZ PAUL LEVINE GEORGETOWN SAMUEL SIMMENS LUCIANE CAVALLISIDNEY FU

DAVID GEFFEN SOMNCIBARBARA JOSEPH PINZONE

VONDERHAAR