Oral Oncology 111 (2020) 104930

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

RUVBL1 is an amplified epigenetic factor promoting proliferation and T

inhibiting differentiation program in head and neck squamous cancers
Derrick Lina,b,1, Brian Linc,1, Haymanti Bhanotd, Rozenn Rioua, Nicholas B. Abta,b,
⁎
Jayaraj Rajagopalc,e, Srinivas Vinod Saladia,b,e,
a
Massachusetts Eye and Ear Infirmary, Harvard Medical School, United States
b
Massachusetts General Hospital Cancer Center, United States
c
Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, United States
d
Dana Farber Cancer Institute, Harvard Medical School, United States
e
Broad Institute of MIT and Harvard, United States

A R T I C LE I N FO A B S T R A C T

Keywords: Mutations in histone modifying enzymes and histone variants were identified in multiple cancers in The Cancer
RUVBL1 (Tip49a) Genome Atlas (TCGA) studies. However, very little progress and understanding has been made in identifying the
RUVBL2 contribution of epigenetic factors in head and neck squamous cell carcinoma (HNSCC). Here, we report the
H2AZ identification of RUVBL1 (TIP49a), a component of the TIP60 histone modifying complex as being amplified and
Head and Neck Squamous Cell Carcinoma
overexpressed in HNSCC. RUVBL1 plays a key role in incorporating histone variant H2AZ in chromatin thereby
(HNSCC)
The Cancer Genome Atlas (TCGA), Cyto-
regulating transcription of key genes involved in differentiation, cancer cell proliferation and invasion. H2AZ is
Keratins also overexpressed in HNSCC tumors thereby regulating RUVBL1/H2AZ dependent transcriptional programs.
Patient data analysis of multiple cohorts including TCGA and single cell HNSCC data indicated RUVBL1 over-
expression as a poor prognostic marker and predicts poor survival. In vitro experiments indicate a pro-pro-
liferative role for RUVBL1/H2AZ in HNSCC cells. RUVBL1 inversely correlates with differentiation program and
positively correlates with oncogenic programs, making it a key contributor to tumorigenesis and a vulnerable
therapeutic target in HNSCC patients.

Introduction of chromosome 3q (harboring p63 and SOX2) is a frequent driver event

in 30% of HNSCC patients in both human papillomavirus (HPV)–posi-
Approximately 50,000 cases of squamous cell carcinoma of the head tive and –negative disease [3–7]. P63 is a master transcription factor of
and neck (HNSCC) are diagnosed annually, while more than 10,000 epithelial development and is also a critical factor for tumor main-
deaths are attributed to this disease. Surgery, radiation and che- tenance in HNSCC [8,9], hence considered a lineage addiction onco-
motherapy are currently the only available treatment options, and little gene in squamous cancers. Although global profiling of p63 identified
progress has been made to increase the overall survival of patients distinct transcriptional programs in normal epithelium and tumors, the
[1,2]. Although genome-wide analyses of many cancer types have re- underlying mechanisms are poorly understood [7]. There are, however,
cently identified activated oncogenes that may be targeted ther- a large number of genes involved in tumor specific transcriptional
apeutically, HNSCCs do not commonly harbor such somatically-mu- programs that involve p63 interaction with additional co-factors. Re-
tated genes. However, activating mutations were identified in cent studies have identified SWItch for Sucrose Non-Fermenting (SWI/
phosphoinositide-3-kinase subunit alpha (PIK3CA) and, Ras family SNF) chromatin remodeling enzyme component, Actin Like 6A
small GTPase, H-Ras, in a subset of HNSCC patients. Epidermal Growth (ACTL6A), as key interacting partner cooperating with p63 in med-
Factor Receptor (EGFR) is overexpressed in majority of HNSCC. How- iating its transcriptional activity. P63 interacts with RuvB like AAA
ever, resistance to EGFR targeted therapies results in modest benefit to ATPase 1/2 (RUVBL1/2), DNA Methyltransferase 1 Associated Protein
patient survival making it important to identify other targeted therapies (DMAP1) and mediates the incorporation of H2AZ at the p63 target
in HNSCC to have better patient outcomes. Additionally, amplification loci, including EGFR, FGFR2, and others. RUVBL1 belongs to ATPases

⁎
Corresponding author at: Massachusetts Eye and Ear Infirmary, Harvard Medical School, United States.
E-mail address: [email protected] (S.V. Saladi).
1
Equal contribution.

https://doi.org/10.1016/j.oraloncology.2020.104930
Received 21 April 2020; Received in revised form 24 June 2020; Accepted 22 July 2020
1368-8375/ © 2020 Published by Elsevier Ltd.
D. Lin, et al. Oral Oncology 111 (2020) 104930

Fig. 1. RUVBL1 alteration in cancer and head and neck squamous cell carcinoma. (A) RUVBL1 is altered across cancer types including amplification, loss and
mutations. (B) RUVBL1 is amplified in 5% of HNSCC. (C) RUVBL1 is overexpressed in 15% and RUVBL2 in 5% of HNSCC cases among TCGA patient samples.

Associated with various cellular activities (AAA+) family. RUVBL1 has specific transcription factor circuitry and the epigenome on super-en-
DNA dependent ATPase activity and DNA helicase activity. RUVBL1/2 hancers and enhancers to identify the driver oncogenes will help in
are components of various protein complexes and contribute to ATP- designing potential therapeutic opportunities in HNSCC.
dependent chromatin remodeling, histone modifications, DNA damage
and repair, and mitosis [10]. RUVBL1 was previously shown to co-
Methods
operate with MYC and β-Catenin in promoting cellular transformation.
H2AZ deposition occurs at distal intergenic regions and gene bodies in
Single cell analysis
cancer cells [11]. P63 binding is mostly at distal intergenic or intronic
enhancer regions in the tumor cells. These observations indicate com-
Processed RNAseq data was accessed from GSE103322 [13]. Sam-
plex chromatin architectural modulation in regulating the transcription
ples were processed in R using RaceID3 algorithms [14], with filtering
of driver oncogenes. Analysis of the tumor specific p63 transcriptional
of minimum 3000 transcripts per cell and a minimum of 5 counts per
programs revealed enrichment of ErbB tyrosine kinase signaling
gene. T-SNE plots were used for dimension reduction and generated in
pathway comprising of Neuregulin 1 (NRG1) and other ErbB signaling
R.
pathway genes including Epidermal Growth Factor Receptor (EGFR).
P63 binds at multiple loci (distal and gene body) and regulates the
expression of NRG1 and EGFR. P63 binding corresponds to dense TCGA analysis
H3K27 acetylation at the bound loci indicating a super-enhancer pro-
file. RUVBL1 was also previously identified as an interacting partner of The Cancer Genome Atlas (TCGA) is a cancer genomics program
p63 in HNSCC. It confers poor prognosis in a subset of HNSCC patients which has molecularly characterized over 30 human cancers, including
where it is amplified, and it complexes with p63 to regulate anti- head and neck squamous cell carcinoma, and their matched normal
apoptotic genes (including Sterile Alpha Motif Domain Containing 9 samples. These data from the National Cancer Institute and the National
Like, (SAMD9L)) to mediate cancer cell proliferation, inhibit apoptosis Human Genome Research Institute are publicly available for analysis.
[12]. Over-expression of RUVBL1 in HNSCC suggests that it might UALCAN [15], which is a portal for facilitating tumor subgroup gene
regulate a subset of genes involved in p63 mediated transcriptional expression along with survival analysis, was utilized to analyze the
reprogramming, particularly differentiation programs. Only 18% of TCGA dataset. Transcriptome data is available for a total of 520 head
patients have been responsive to immunotherapy. Delineating tumor and neck squamous cell carcinoma patients. There was > 10 years
follow up time available for all data points. RUVBL1 and RUVBL2

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Fig. 2. RUVBL1 is overexpressed in tumors. (A) RUVBL1 & 2 are over expressed in TCGA HNSCC tumors (n = 520) compared to normal epithelium (n = 44). (B)
RUVBL1 is overexpressed in an independent cohort (Peng’s) of squamous cell carcinoma tumors. (C) TCGA analysis indicates correlation of amplification to increased
gene expression of RUVBL1. (D).

mRNA expression levels were compared amongst normal head and neck revealed amplification of RUVBL1 in 5% of HNSCC patients whereas its
squamous epithelium and HNSCC. Cbioportal was utilized to analyze interacting partners were amplified in only a few cases (0.8%) (Fig. 1b).
amplification and overexpression of RUVBL1, RUVBL2 and H2AZ in RNA-seq analysis of the TCGA patient cohort revealed overexpression of
HNSCC patients. RNASeq from TCGA data was utilized to do the cor- RUVBL1 in 16% of patients and RUVBL2 in 5% of patients (Fig. 1c).
relation analysis [16,17]. Whole transcriptome analysis of patient samples showed that the ex-
pression of RUVBL1 and RUVBL2 is significantly higher in tumors
Plasmids, cell lines and knockdown compared to normal adult epithelium (Fig. 2a). Additional analysis of
patient cohorts including Peng’s head and neck cohort (n = 79), and
shRNA against RUVBL1 and H2AZ were obtained from the mole- Talbot lung (n = 93) indicate overexpression of RUVBL1 in squamous
cular profiling laboratory at MGH. shRNA sequences are provided in tumors (Fig. 2b). RUVBL1 interacting partner, RUVBL2 is expressed at a
table 3. BICR22 cell line was used for the knockdown experiments. basal level and not amplified in HNSCC. Gene expression analysis of
Lentiviral production was performed in 293 T cells. Viral infection was multiple HNSCC cell lines including the immortalized normal epithelial
performed as reported previously [7]. cell line HaCaT, used as a control cell line, showed high expression of
RUVBL1 in tumor cell lines with BICR22 showing the highest expression
Colony formation assay (Fig. 2c). We further analyzed and identified mRNA expression levels of
RUVBL1 correlating to the copy number variation in HNSCC patients
HNSCC cells were infected with shRNA against RUVBL1 and H2AZ. compared to patients with gain, shallow deletion, and loss (Fig. 2d).
10,000 cells were seeded per well in a 6 well plate after infection. Two
weeks after infection, cells/colonies were stained with crystal violet to H2AZ is overexpressed in HNSCC tumors
assess the colony forming capacity of the cells.
RUVBL1 and RUVBL2 function by promoting the incorporation of
Results histone variant H2AZ at t + 1 Transcription Start Sites (TSS) of target
promoters [18]. Incorporation of H2AZ helps in remodeling of chro-
RUVBL1 is amplified in HNSCC matin and regulation of gene expression [18–20], affecting various
genes including Cyclin D1 [20], and SAMD9L, an anti-apoptotic gene in
TCGA analysis (n = 530) revealed amplification of RUVBL1 in SCC’s [12]. Since we observed amplification and increase in gene ex-
multiple cancers including squamous cell carcinomas of lung, head and pression of RUVBL1, we wanted to investigate the levels of H2AZ. We
neck and esophagus (Fig. 1a). Further analysis of head and neck found overexpression of H2AZ in TCGA data set (Fig. 2e) as well as the

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Fig. 3. Single cell analysis of RUVBL1, 2 and H2AZ. (A, C, E) Violin plots indicating enrichment of RUVBL1, 2 and H2AZ in tumor cells compared to immune or
stromal cells in HNSCC. (B, D, F) T-SNE plots of single cell RNAseq HNSCC.

Table 1 Single cell analysis of RUVBL1/RUVBL2/H2AZ mRNA expression

Inverse correlation of RUVBL1 with differentiation program.
Gene Spearman’s Correlation P-Value Q-Value Bulk tumors were utilized for TCGA studies. Recent studies have
highlighted the importance of heterogeneity in tumors as a mechanism
KRT2 −0.282 1.64e−10 1.54e−9 by which tumors bypass current treatment options. Single Cell Analysis
KRT6C −0.187 2.905e−5 1.012e−4 (SCA) was recently performed on head and neck squamous tumors [13].
KRT6A −0.162 3.044e−4 8.510e−4
KRT1 −0.153 6.461e−4 1.683e−3
We utilized this single cell data and performed expression analysis of
IVL −0.15 7.748e−4 1.982e−3 RUVBL1 and RUVBL2. RUVBL1 was overexpressed at mRNA level,
KRT5 −0.129 4.084e−3 8.832e−3 ubiquitously in all tumor cells, but very few stromal and immune cells
KRT13 −0.0939 0.0366 0.06 (Fig. 3a, b). RUVBL2 was also overexpressed in all the tumor cells and a
significant portion of stromal cells but not the immune cells (Fig. 3c, d).
We found selectively high expression of RUVBL1 and RUVBL2 in
Peng’s cohort (Fig. 2f). This indicates that there is no limiting step to
tumor cells (Fig. 3a, c). Furthermore, the effector gene H2AZ was highly
amplification and overexpression of RUVBL1 in patients as there is
expressed in tumor cells (Fig. 3e). H2AZ is an essential histone variant
overexpression of the effector gene, H2AZ, which is required for the
expressed ubiquitously in cells. Interestingly H2AZ was expressed more
transcriptional regulation of genes mediated by RUVBL1.
in the immune cells (Fig. 3f), which validates recent observation where
H2AZ was shown to modulate the transcriptional program of immune
cells [21]. Overexpression of H2AZ in immune cells along with tumor
cells indicating its importance in regulation of gene expression in

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Fig. 4. Inverse correlation of RUVBL1 to differentiation program. (A, B, C) RUVBL1 expression correlates inversely to differentiation genes indicated here, KRT10, 6C,
IVL which includes both early and late differentiation markers. (D) SAMD9L, anti-proliferative gene inversely correlates to RUVBL1 in HNSCC tumors. (n for this
analysis = 520).

Fig. 5. Overexpressed RUVBL1 correlates to poor

disease free survival. (A) Patients with high ex-
pression of RUVBL1 have poor disease free sur-
vival. Red-high (n = 63) and blue-low (n = 316)
expression of RUVBL1 as assessed from the TCGA
data. (B) Schematic showing RUVBL1 functions
through multiple factors including TRRAP and
promotes transformation, tumorigenesis.

HNSCC immune cells. Established markers for basal cells (Keratin 5), RUVBL1 inversely correlates to differentiation program in HNSCC
stromal cells (ACTA2) and immune cells (PRF1) were used in the single
cell analysis to stratify the different cell types (supplementary). ACTL6A, an interacting partner of RUVBL1/2 and SWI/SNF chro-
matin complexes, is amplified in HNSCC and regulates regenerative
proliferation program and suppresses differentiation. Early and late
differentiation markers were previously shown to be suppressed by

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Fig. 6. H2AZ expression correlates inversely to differentiation program in HNSCC. (A, B, C) H2AZ expression correlates inversely to differentiation genes involved in
HNSCC as indicated here, KRT1, 6C, 14 which includes both early and late differentiation markers and (D) inversely correlates to SAMD9L in HNSCC tumors. (n for
this analysis = 520).

Table 2 samples (Fig. 4d). These observations indicate that RUVBL1 over-
Inverse correlation of H2AZ with differentiation program. expression promotes an anti-proliferative state and suppresses the dif-
Gene Spearman’s Correlation P-Value Q-Value
ferentiation program thereby maintaining oncogenic state in these tu-
mors.
KRT1 −0.392 1.03e−19 1.79e−18
KRT6C −0.384 7.42e−19 1.15e−17
KRT6A −0.379 2.10e−18 3.13e−17
Prognostic value of RUVBL1 in tumors
KRT10 −0.302 6.56e−12 4.37e−11
KRT14 −0.274 5.61e−10 2.97e−9 Previously, ACTL6A amplification was shown to correlate to poor
IVL −0.309 1.93e−12 1.37e−11 prognosis in head and neck cancer patients. Similarly, here we identify
LOR −0.152 7.069e−4 1.57e−3
the prognostic value of RUVBL1 in HNSCC patients. Analysis utilizing
the data obtained from TCGA data indicated poorer disease-free sur-
vival in patients with overexpression of RUVBL1 (16%) compared to
ACTL6A in HNSCC. We analyzed TCGA data to investigate the corre-
patients with low expression of RUVBL1 (Fig. 5a). RUVBL1, as a core
lation of RUVBL1 expression to differentiation. RNA analysis of the
component of Transformation/Transcription Domain-Associated Pro-
TCGA data revealed inverse correlations to multiple differentiation
tein (TRRAP) complex, promotes deposition of H2AZ at target gene
markers including early differentiation markers Keratin 1, 10, 14, as
promoters and enhancers, regulating target gene expression, thereby
well as late differentiation markers Involucrin (IVL), indicating a strong
contributing to transformation of cells or promote tumorigenesis
suppression of differentiation program by RUVBL1 in head and neck
(Fig. 5b).
tumors (Table 1). Further investigation revealed strong inverse corre-
lations of RUVBL1 to multiple differentiation markers including KRT10,
KRT6C and IVL (Fig. 4a–c). Previously, RUVBL1 was shown to strongly H2AZ as an effector of RUVBL1 in regulating differentiation program
suppress the transcription of SAMD9L, an anti-apoptotic gene, through
incorporation of H2AZ in a p63 dependent manner in head and neck RUVBL1 functions by incorporating H2AZ at the target promoters.
squamous cancer cells [12], indicating an inverse correlation of ex- We observed inverse correlation of RUVBL1 to key differentiation genes
pression in tumors. As expected, we observed a strong inverse corre- including KRT1, KRT6C, and KRT14 (Fig. 6a–c), and anti-apoptotic
lation of expression of SAMD9L to RUVBL1 in HNSCC patient tumor gene, SAMD9L (Fig. 6d). Accordingly, we also found a strong inverse
correlation of H2AZ with other early and late differentiation markers

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Fig. 7. RUVBL1 and H2AZ suppress differentiation and promote proliferation. (A, B) Knocking down RUVBL1 and H2AZ results in increased expression of differ-
entiation genes and anti-proliferation gene CDKN1A/p21. (C, D) Knocking down RUVBL1 and H2AZ results in decreased proliferation as assessed by colony formation
assay.

including KRT10, IVL and LOR (Table 2), in HNSCC tumors, collectively and overexpressed in subset of HNSCC tumors. One of the major in-
indicating the involvement of H2AZ in regulating differentiation. Fi- novations of this study is the dissection of the deregulated epigenome,
nally, we also observed a strong inverse correlation to SAMD9L, a which will allow an unprecedented understanding of the complexity of
previously reported target of RUVBL1/H2AZ in HNSCC. transcription and epigenetic factor mediated transcriptional repro-
gramming in HNSCC. Global binding analysis of p63 in HNSCC cells
revealed enrichment at distal enhancers (intergenic regions ~45%,
RUVBL1 and H2AZ regulate genes involved in differentiation and
intronic regions ~45%) than at transcription start sites (10%) [7]. We
proliferation
expect that this new knowledge will allow us to identify novel driver
oncogenes that are druggable targets. Epigenetic deregulation in
We wanted to test if RUVBL1 and H2AZ regulate gene expression
HNSCC is poorly understood and this study identifies a p63/RUVBL1/
involved in differentiation. To test this, we utilized shRNA against
H2AZ axis, where in RUVBL1 is amplified in subset of patients, and
RUVBL1 and H2AZ and found that knocking down RUVBL1 (Fig. 7a) or
modulates target genes involved in differentiation and proliferation.
H2AZ (Fig. 7b) resulted in increase in KRT6A/C and KRT5, and late
This will have therapeutic value in the early and invasive stages of
differentiation marker Involucrin. Furthermore we did colony formation
HNSCC, potentially leading to the development of drugs targeting
assay to analyze the effect of RUVBL1 and H2AZ on HNSCC cell pro-
tumor specific programs, thereby controlling this aggressive disease.
liferation. Knocking down RUVBL1 and H2AZ resulted in decreased
p63 is amplified and overexpressed in the majority of HNSCC cases.
number of colonies, indicating role of RUVBL1 and H2AZ in prolifera-
RUVBL1, an interacting partner of p63, is also amplified in a subset of
tion (Fig. 7c).
HNSCC patients. P63 through interaction with RUVBL1 mediates de-
position of H2AZ at p63 target genes including SAMD9L and other
Discussion genes. Deposition of H2AZ at the target loci followed by histone acet-
ylation results in either activation or repression of target genes in a
Much of the focus in TCGA studies is identification of constitutive context dependent manner [22]. Over-expression of H2AZ in immune
activating mutation of signaling pathway genes and epigenetic factors, cells as observes in single cell analysis indicates its importance in reg-
however very little focus has been on the amplification of epigenetic ulating the expression of genes in the immune cells which needs to be
factors. HNSCC unlike lung cancer has very few driver oncogenes further explored. Multiple studies have identified epigenetic factors and
identified so far and majority of mutations in genes identified so far are histone modifiers that interact with p63 and contribute to HNSCC.
inactivating mutations. Our own recent study has identified ACTL6A, RUVBL1, along with RUVBL2, previously identified as interacting
subunit of SWI/SNF complex as an amplified driver oncogene in partners of p63, are which are involved in many cell processes im-
HNSCC. ACTL6A is present on the 3q chromosome. The 3q amplicon perative for oncogenesis [23,24]. These have been found to be
also harbors ACTL6A interacting partner, RUVBL1 which is amplified

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