PHF21B as a candidate tumor
suppressor gene in head and neck
squamous cell carcinomas
Silvia R Rogatto
Faculty of Medicine – Botucatu – SP
AC Camargo Cancer Center – SP – BRAZIL
[email protected]
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 O
Ferreira, Ademar Lopes, Eduardo Abrantes, Eduardo N Pereira Lima,
Antonio Hugo M Campos, Diogo F C Patrão, Alex Fiorino, Erika M Monteiro
Santos, José Vassalo, Samuel Aguiar Junior, Carla Rosenberg, Ana Cristina
Krepischi,, Eduardo Moraes Reis, Aline Silva, Maria do Rosário D.O. Latorre,
Cristovam Scapulatempo, Alexandra Brentani, Ana Luiza Viana, Paulo E
Mangeon Elias, Eliana B de Araujo, André Lopes Carvalho, Edmundo
Mauad, Luciano Souza Viana, Marcos Duarte de Mattos, José R Fígaro
Caldeira, Rogério H Saad, Maria A Custódio Domingues, Patricia Pintor dos
Reis, Claudia A Rainho, Marcos Venício Alves Lima, Sérgio Joaçaba, Rosane
Santana, Ilce M de Syllos Cólus, Emmanuel Dias Netto, Diana Noronha
Nunes, Fernando Augusto Soares, Maria Mitzi Brentani, Vilma Regina
Martins, Glaucia N. Hajj, Tiago G. dos Santos, Michele Christine
Landemberger, Elisa Napolitano Ferreira, Bianca Garcia Lisboa, Rafael
Malagoli, 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-Fraumeni
Syndrome, Familial Adenomatous Polyposis and
Familial Melanoma
Exome sequencing, RNAseq and
Methylome: sporadic and hereditary
tumors - breast , colorectal, head neck,
thyroid, melanoma
TOTAL - 985
Hallmarks of Hereditary Cancer Predisposition
Syndromes
Younger
patients
Family history
of cancer: with
the same or
related types of
cancer
Multiple
primary cancer
HNSCC no
alcohol and
tobacco users
5-10% cases: Hereditary predisposition
2005
D22S273 –
22q13.3
qPCR: loss
Family history
of HNC
OR = 42.6 for
current
smokers, heavy
drinkers with
family history
2008
OR = 7.2 among
subjects with
family history,
alcohol and
tobacco users
2009
NM_138415
PHF21B
13 exons – PHD
zinc finger domain
22q13.3 deletion
Shorter survival
Family history
association
2014
Bertonha et al, 2014
Reis et al, 2002b
Reis et al, 2002
LOH in 22q in
HNC
Putative TSG
2002
Bergamo et al, 2005
1995
Garavello et al, 2008
Lange et al, 2002
Yu et al, 2002
RR=1.97
Foulkes et al, 1995
Cooper et al 1995
RR=3.5
TP16 mutations
in two families
with HNSCC
Familial
factors play a
role in the
etiology of
HNC.
Negri et al, 2009
1st degree relatives
HNSCC
History of oral and
pharyngeal ca and
laryngeal ca is a
strong determinant
of oral and
pharyngeal ca risk,
independent from
tobacco and alcohol.
PHF21B as a putative tumor supressor gene
PHF21B encodes the PHD finger protein 21B, composed of 531 amino acids
(NP_612424.1) with no previously established function
Eligibility criteria
absence of previous histological
diagnosis of any cancer type
tumor larger than 1 cm in size
availability of complete
clinicopathological data
Exclusion Criteria
Lip, nasopharynx, thyroid and salivary gland
tumors
patients having systemic predisposing
diseases, such as Epidermolysis bullosa,
Xeroderma pigmentosum, Juvenile
papillomatosis and Fanconi’s anemia.
Familial history of
cancer
at 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:
bladder,
uterine/cervical
melanoma
breast,
stomach,
(Negri et al., 2009).
kidney,
cancers
urinary
and
skin
75 HNC patients
* 1st degree relatives
40 family history
cancer
18: at least 2
affected relatives
by HNC
10: at least 1
tobacco associated
tumor in affected
relatives
Other cancer sites:
5 colon*
1 Thyroid*
2 Pancreas*
1 Prostate*
1 Lymphoma
3 Breast*
2 Skin cancer*
10: 1 affected
relative with other
tumor type than
HNC.
7 had at least 1 first
degree relative
HNC
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.
transcript variant 1
NM_138415.4
NM_001135862.2
transcript variant 2
transcript variant 3
NM_001242450.
The PHF21B gene and its flanking regions are highly GCrich.
This island spans 3336 bp, including the 5' region, with the first
two exons of this gene overlapping three alternative splicings.
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
Breast cancer cell lines
HNSCC cell lines
Pharmacologic
DNA methylation
inhibition:
High expression
levels after 5-AzadC in 2 cell lines
Colon cancer cells
Effect of epigenetic
Biological inhibition:
treatment with 5-Aza-dC
on PHF21B gene
 DNA methylation in HCT116 cells was inversely
expression
correlated with PHF21B gene
 DKO cells (unmethylated) expressed the gene
PHF21B
50%
0%
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
PHF21B transcripts potentially produce several
proteins with no sequence overlapped
Adapted from Lallous N & Ramón-Maiques S (2011)
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
2009/50262-6
2012/ 04370-4
Brazilian National Institute of Science and Technology in Oncogenomics
(FAPESP 2008/57887- 9 and CNPq 573589/08-9)