Silvia R RogattoFaculty of Medicine – Botucatu – SP

AC Camargo Cancer Center – SP – BRAZIL

rogatto@fmb.unesp.br

PHF21B as a candidate tumor

suppressor gene in head and neck

squamous cell carcinomas

National Institutes of Science and Technology - INCT

TOTAL:122

SP: 44

INCITO - National Institute of Science

and Technology in Oncogenomics

Luiz Paulo Kowalski, Sergio Verjovsky-Almeida, Silvia Regina Rogatto,Dirce M Carraro, Helena P Brentani, Maria Izabel W Achatz, Fabio OFerreira, Ademar Lopes, Eduardo Abrantes, Eduardo N Pereira Lima,Antonio Hugo M Campos, Diogo F C Patrão, Alex Fiorino, Erika M MonteiroSantos, José Vassalo, Samuel Aguiar Junior, Carla Rosenberg, Ana CristinaKrepischi,, Eduardo Moraes Reis, Aline Silva, Maria do Rosário D.O. Latorre,Cristovam Scapulatempo, Alexandra Brentani, Ana Luiza Viana, Paulo EMangeon Elias, Eliana B de Araujo, André Lopes Carvalho, EdmundoMauad, Luciano Souza Viana, Marcos Duarte de Mattos, José R FígaroCaldeira, Rogério H Saad, Maria A Custódio Domingues, Patricia Pintor dosReis, Claudia A Rainho, Marcos Venício Alves Lima, Sérgio Joaçaba, RosaneSantana, Ilce M de Syllos Cólus, Emmanuel Dias Netto, Diana NoronhaNunes, Fernando Augusto Soares, Maria Mitzi Brentani, Vilma ReginaMartins, Glaucia N. Hajj, Tiago G. dos Santos, Michele ChristineLandemberger, Elisa Napolitano Ferreira, Bianca Garcia Lisboa, RafaelMalagoli, Martín Roffé, Sandra D. Linde , João Duprat P. Neto

Hereditary Cancer Syndromes and Familial Aggregation of Cancer

Screening of mutations and copy number variations (CNVs) in Hereditary Cancer Syndrome and Familial Aggregations: Colorectal Cancer - Lynch Syndrome,

Breast and Ovarian Carcinoma, Li-FraumeniSyndrome, Familial Adenomatous Polyposis and

Familial Melanoma

Exome sequencing, RNAseq and Methylome: sporadic and hereditary

tumors - breast , colorectal, head neck, thyroid, melanoma

TOTAL - 985

5-10% cases: Hereditary predisposition

Younger patients

Family history of cancer: with

the same or related types of

cancer

Multiple primary cancer

HNSCC no alcohol and

tobacco users

Hallmarks of Hereditary Cancer Predisposition Syndromes

1995 2014

Re

is e

t a

l, 2

00

2

Co

op

er e

t al

19

95

Fou

lkes

et

al, 1

99

5

Lan

ge e

t al

, 20

02

Yu

et

al, 2

00

2

Gar

avel

lo e

t al

, 20

08

Neg

ri e

t al

, 20

09

Familial

factors play a

role in the

etiology of

HNC.

TP16mutations in two families with HNSCC

Re

is e

t a

l, 2

00

2b

D22S273 –22q13.3qPCR: lossFamily history of HNC

PHF21B as a putative tumor supressor gene

LOH in 22q in HNCPutative TSG

1st degree relativesHNSCC

RR=3.5 RR=1.97

2002 2008

History of oral and pharyngeal ca and laryngeal ca is a strong determinant of oral and pharyngeal ca risk, independent from tobacco and alcohol.

20092005

Be

rga

mo

et

al,

20

05

22q13.3 deletionShorter survivalFamily history association

Be

rto

nh

a e

t a

l, 2

01

4

OR = 42.6 for

current

smokers, heavy

drinkers with

family history

OR = 7.2 among subjects with family history, alcohol and tobacco users

NM_138415 PHF21B

13 exons – PHD zinc finger domain

PHF21B encodes the PHD finger protein 21B, composed of 531 amino acids

(NP_612424.1) with no previously established function

Eli

gib

ilit

y c

rite

ria

absence of previous histological diagnosis of any cancer type

tumor larger than 1 cm in size

availability of complete clinicopathological data

Exc

lusi

on

Cri

teri

a

Lip, nasopharynx, thyroid and salivary gland tumors

patients having systemic predisposing diseases, such as Epidermolysis bullosa,

Xeroderma pigmentosum, Juvenile papillomatosis and Fanconi’s anemia.

Fa

mil

ial

his

tor

y o

f c

an

ce

rat least 2 first-degree relatives in the family affected

with HNC and/or other related cancers

age of cancer onset lower than 45 years old in at least one of the affected family members and/or the

proband

any age at onset when the HNC patient reported no tobacco and/or alcohol consumption or other

related well-known etiological factor

HNSCC-associated tumors: stomach, kidney, urinary

bladder, breast, uterine/cervical cancers and skin

melanoma (Negri et al., 2009).

75 HNC patients

18: at least 2 affected relatives

by HNC

10: at least 1 tobacco associated tumor in affected

relatives

10: 1 affected relative with other

tumor type than HNC.

7 had at least 1 firstdegree relative

HNC

40 family history cancer

Other cancer sites:5 colon*1 Thyroid*2 Pancreas*1 Prostate*1 Lymphoma3 Breast*2 Skin cancer*

* 1st degree relatives

Association between chromosome 22 deletions and

family cancer history based on the two- hit hypothesis

of tumor suppressor inactivation

To investigate potential mechanism of gene inactivation: SANGER SEQUENCING

PHF21B mutations were assessed in 26 paired samples (normal and tumor)

Exons 6, 7, and 8, encoding for the PHD

domain

Exons 3, 9 ,10 and 11

The absence of mutations in the PHD domain of the PHF21B

gene are in accordance with the literature for other genes

encoding proteins containing the same domain, such as the

inhibitor of growth (ING) family.

Based on this result: the promoter methylation, could

be associated with PHF21B down-regulation.

The PHF21B gene and its flanking regions are highly GC-

rich.

This island spans 3336 bp, including the 5' region, with the first

two exons of this gene overlapping three alternative splicings. tr

ansc

rip

t v

aria

nt

1

NM

_1

38

41

5.4

tran

scri

pt

var

ian

t 2

NM

_0

011

35

86

2.2

tran

scri

pt

var

ian

t 3

NM

_0

01

24

24

50

.

Characterization of promoter-associated CpG island of PHF21B

REGION 1 overlaps and is associated with an active promoter as suggested by:

1. the enrichment of histone marks (H3K4me3 and H3K27Ac)

2. DNaseI hypersensitive site

3. transcription factor binding sites

4. Pol2 occupancy

Effect of epigenetic

treatment with 5-Aza-dC

on PHF21B gene

expression

Pharmacologic

DNA methylation

inhibition:

High expression

levels after 5-Aza-

dC in 2 cell lines

Breast cancer cell lines HNSCC cell lines

DNA methylation in HCT116 cells was inversely

correlated with PHF21B gene

DKO cells (unmethylated) expressed the gene

PHF21B

Biological inhibition:

Colon cancer cells

0%

50%

Subcellular distribution of endogenous and transfected PHF21B:

Endogenous and transfected PHF21B localize to the nucleus: transcriptional control

Cellular resistance to cisplatin

Overexpression of PHF21B reduced the migratory ability

Homo sapiens complex locus PHF21B, encoding PHD finger protein 21B

PHD motif recognizes methylated lysines

in histone N-terminal polypeptides

Adapted from Lallous N & Ramón-Maiques S (2011)

PHF21B transcripts potentially produce several

proteins with no sequence overlapped

Proteins are expected to have molecular functions

(metal ion binding, protein binding, histone

mark reader, zinc ion binding) and to localize in

nucleus

No phenotype has yet been reported to our

knowledge

in vivo function is yet unknown

Brazilian National Institute of Science and Technology in Oncogenomics

(FAPESP 2008/57887- 9 and CNPq 573589/08-9)

2009/50262-6

2012/ 04370-4