ACTIVATION OF BP1 IS ASSOCIATED WITH AGGRESSIVE BREAST CANCER PATRICIA BERG, PH.D.

pBP1 IS AN ISOFORM OF THE DLX4 GENE CHASE ET AL, MOL. CELL. BIOL. 22: NOTE: BP1 AND DLX4 ARE NOT INTERCHANGEABLE TERMS BP1 DLX7 DLX4

CHARACTERISTICS 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 BP1 (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 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 (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 Atlas Genet Cytogenet Oncol Haematol. 15:

GENES INVOLVED IN NON- HEREDITARY BREAST CANCER GENEFREQUENCY HER-220 – 30% C-MYC20 – 30% CYCLIN E30% EGFR25-35% 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 0.03 Negative0 (0%) 18 (100%) Positive7 (27%) 19 (73%) PR Status 0.02 Negative1 (4.0%) 24 (96%) Positive6 (33%) 12 (67%) Race 0.04 Caucasian6 (43%) 8 (57%) African American3 (11%) 25 (89%) FU et al, Br. Ca. Res. 5:

BP1 AND BREAST CANCER PROGRESSION MAN ET AL, BR. CA. RES. & TREAT. 90:

BP1 and Progression NORMAL 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

SUMMARY 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 CANCER 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 CANCER 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, Ca. Biomarkers 5: 9-17.

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

HOW IS BP1 ACTIVATED? A. DNA AMPLIFICATION B. ESTRADIOL

A. 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, ** FU ET AL, GENE 278:

FISH analysis of FFPE sections of a primary breast tumor (left) and SLN metastasis (right) from one patient showing amplification of the BP1 gene FISH ANALYSIS OF BP1

CORRELATION BETWEEN BP1 AMPLIFICATION AND EXPRESSION ALL CASES SHOWING BP1 AMPLIFICATION ALSO SHOWED BP1 EXPRESSION BY IHC BP1 POSITIVITY WAS ALSO SEEN IN CASES WITHOUT AMPLIFICATION

BP1 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 ChIP 1. LADDER 2.3 INPUT CONTROL FOR ERE ELEMENT IN EXON I AND INTRON I 4.5 AFTER IP WITH ERα AB

BP1 ACTIVATES ONCOGENES GENEASSOCIATED WITH MMP9INITIATION, INVASION, METASTASIS BCL-2ANTI-APOPTOTIC (Stevenson et al, 2007) C-MYCPROLIFERATION, DIFFERENTIATION, APOPTOSIS (Trinh et al, 2011) MET VEGF GROWTH, ANGIOGENESIS, METASTASIS ANGIOGENESIS (Hara et al, 2007)

BP1 EXPRESSION IS ASSOCIATED WITH AGGRESSIVENESS

MEASURES OF AGGRESSIVENESS 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 D4D13 GROWTH IN SOFT AGAR

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

EFFECT OF BP1 ON TUMOR GROWTH IN MICE

ESTROGEN INDEPENDENT TUMOR FORMATION IN MICE Cell Type InjectedEstrogen added Absence of Estrogen MCF-7/EV-14/10 (40%)0/10 (0%) MCF-7/BP1-210/15 (67%)2/10 (20%) MCF-7/BP1-47/11 (64%)2/10 (20%)

BP1 UPREGULATES ERα BOTH DIRECTLY AND INDIRECTLY SUBMITTED

DIRECT: BP1 ACTIVATES ERα RNA BY BINDING TO IVS1

INDIRECT: BP1 STABILIZES ERα BY UPREGULATING p300 (p300 ACETYLATES ERα) p300 ACTIN O1 V1 mRNA PROTEIN

INDIRECT: 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

MODEL

CONCLUSIONS BP1 EXPRESSION MAY BE PROGNOSTIC IN BREAST CANCER BP1 IS A NEW POTENTIAL THERAPEUTIC TARGET

ACKNOWLEDGEMENTS GWUMC SAURABH KIROLIKAR JHU SANKET AWATE JUDITH KARP DANIEL RHEEY FARHAD VESUNA BIN JIN HUANG VENU RAMAN KELLIE YAMANE YASSI FALLAH AFIP YAN-GAO MAN ARNOLD SCHWARTZ PAUL LEVINE GEORGETOWN SAMUEL SIMMENSLUCIANE CAVALLI SIDNEY FU DAVID GEFFEN SOM NCI BARBARAJOSEPH PINZONE VONDERHAAR