ikkβ-mediated resistance to skin cancer development is ......2017/06/02 · 1 ikkβ-mediated...

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IKKβ-mediated Resistance to Skin Cancer Development

is Ink4a/Arf-dependent

Angustias , 2 ,3, Ana Bravo 4, Cristian Suarez-Cabrera 1, 2, 3, Josefa P.

Alameda 1, 2, 3, M. Llanos Casanova 1, 2, 3, Corina Lorz 1, 2, 3, Carmen Segrelles 1,

2, 3, José C. Segovia 5, 6, Jesús M. Paramio 1, 2, 3, Manuel Navarro 1, 2, 3, Angel

Ramirez 1, 2, 3

1 Molecular Oncology Unit. Centro de Investigaciones Energéticas,

Medioambientales y Tecnológicas (CIEMAT), 28040 Madrid, Spain.

2 Oncogenomic Unit, Institute of Biomedical Investigation “12 de Octubre i+12”,

28041 Madrid, Spain.

3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC).

4 Department of Anatomy, Animal Production and Veterinary Clinical Sciences,

Faculty of Veterinary Medicine, University of Santiago de Compostela, 27002

Lugo, Spain.

5 Hematopoietic Innovative Therapies Division. Centro de Investigaciones

Energéticas, Medioambientales y Tecnológicas (CIEMAT). Centro de

Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER).

28040 Madrid, Spain.

6 Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-

Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain.

Running title: Anti-tumoral Activity of IKKβ in Skin

Keywords: IKKβ, p16, p19, non-melanoma skin cancer, Squamous Cell

Carcinoma, Spindle SCC.

on May 14, 2021. © 2017 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 5, 2017; DOI: 10.1158/1541-7786.MCR-17-0157

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Abbreviations list: BrdU: bromodeoxyuridin; DMBA: 7, 12-Dimethylbenz [a]

anthracene; FBS: fetal bovine serum; H&E, hematoxylin and eosin; NF-κB:

nuclear factor kappa B; NMSC: Non-melanoma skin cancer; SCC: Squamous

Cell Carcinoma; SpSCC: spindle cell SCC; Tg, transgenic; TPA: 12-O-

tetradecanoylphorbol-13-acetate; TSG: tumor suppressor gene; wt, wild type.

Financial support: This research was supported partially by funds from Fondo

Europeo de Desarrollo Regional (FEDER) and by grants from the Spanish

government (PI14/01403 to A. Ramírez; PI16/00161to M.L. Casanova;

SAF2015-66015-R, ISCIII-RETIC RD12/0036/0009, PIE 15/00076, and

CB/16/00228 to J.M. Paramio ; and RD16/0011/0011, SAF2014-54885-R, and

RTC2015-3393-1 to J.C. Segovia).

Corresponding author: Angel Ramirez.

E-mail address: [email protected]

mailing address: CIEMAT, ED 70

Avda. Complutense 40

28040 Madrid. Spain.

Phone: 34 91 346 0882; Fax: 34 91 346 6484.

Total number of figures and tables: 4 Figures and 2 Tables.

Supplementary material: 5 Supplementary Figures, 1 Supplementary Table

and a document with the legends to Supplementary Figures and Supplementary

Material and Methods.

Implications: The ability of IKKβ to promote or prevent carcinogenesis

suggests the need for further evaluation when targeting this protein.




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Abstract

IKKβ (encoded by IKBKB) is a protein kinase that regulates the activity of

numerous proteins important in several signaling pathways, such as the nuclear

factor kappa B (NF-κB) pathway. IKKβ exerts a pro-tumorigenic role in several

animal models of lung, hepatic, intestinal and oral cancer. In addition, genomic

and proteomic studies of human tumors also indicate that IKBKB gene is

amplified or overexpressed in multiple tumor types. Here, the relevance of IKKβ

in skin cancer was determined by performing carcinogenesis studies in animal

models overexpressing IKKβ in the basal skin layer. IKKβ overexpression

resulted in a striking resistance to skin cancer development and an increased

expression of several tumor suppressor proteins, such as p53, p16 and p19.

Mechanistically, this skin tumor-protective role of IKKβ is independent of p53

but dependent on the activity of the p16INK4A/p19ARF locus. Interestingly, in

the absence of p16 and p19, IKKβ increased expression favors the appearance

of cutaneous spindle cell-like squamous cell carcinomas, which are highly

aggressive tumors. These results reveal that IKKβ activity prevents skin tumor

development, and shed light on the complex nature of IKKβ effects on cancer

progression, as IKKβ can both promote and prevent carcinogenesis depending

on the cell type or molecular context.




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Introduction

Cancer is a multifactorial disease, caused by alterations in oncogenes that

activate growth-promoting signaling cascades or in tumor suppressor genes

whose inactivating mutations lead to DNA reparation defects, genomic

instability or defective control of cell proliferation. Multiple studies indicate that

deregulation of the activity of NF-κB can result in cancer development. NF-κB is

a conserved family of ubiquitous transcription factors that regulates the

expression of many genes involved in cell proliferation, survival, apoptosis and

other essential processes, which is usually over activated in inflammatory

diseases and in cancer [reviewed in (1)]. NF-κB is usually bound to inhibitory

proteins of the I-κB family, which upon activation are phosphorylated by the IKK

complex and degraded by the proteasome; consequently, NF-κB is released

and modifies the expression of a myriad of genes containing specific DNA

regulatory elements.

The multiprotein complex IKK is formed by the regulatory subunit IKKγ and the

catalytic subunits IKKα and IKKβ. The first known function of IKKβ kinase was

the regulation of NF-κB activation upon proinflammatory stimuli, but it also

performs important NF-κB-independent functions, as IKKβ phosphorylates a

plethora of target proteins, thus controlling their activity and stability. Important

tumor suppressor genes (TSGs), like p53 (2) and p16 (3) are targets of IKKβ;

the same holds true for other relevant proteins in cancer (as MDM2, p63 family

members, ATM, IRS-1, TSC-1, or FOXO3a [reviewed in (4-6)]). Furthermore,

studies performed in MCF-7 mammary gland cells revealed the astonishing

variety of IKKβ substrates, as more than 4000 proteins containing

phosphorylation sites for IKKβ were found (7). In this way, IKKβ affects mTOR,




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insulin and Wnt signaling, immune responses, autophagy, response to DNA

damage and cell transformation. In summary, IKKβ acts over many different

cellular processes, including tumoral transformation, both dependently and

independently of NF-κB.

Animal models with altered expression of members of the NF-κB signaling

pathway highlight the importance of these proteins in skin cancer. For example,

mice lacking p65, the main NF-κB subunit, are resistant to skin carcinogenesis

(8); the expression in proliferative skin cells of a super repressor form of I-κBα

leads to hyperplasia, to a strong inflammatory response and finally to the

development of squamous cell carcinomas (SCCs) (9); IKKα also has a role in

skin cancer, showing protumoral or tumor-suppressive activity depending on the

experimental setting (10) (11).

Regarding the relationship between IKKβ and cancer, a tumor promoting activity

of IKKβ in intestinal, hepatic, lung and pancreatic cancer has been reported (12-

17). In myeloid cells, by contrast, IKKβ is implicated in orchestrating an

antitumoral immune response against melanoma cells (18). So, although IKKβ

promotes tumor development in general, its actual effect over tumor growth and

malignancy is cell type specific. We have previously described that IKKβ

promotes tumor development in oral epithelia (19), but its role in skin cancer

has not been determined yet. In this work, we explore the effect of IKKβ in skin

cancer and the underlying molecular pathways by using transgenic mice with

increased levels of epidermal IKKβ and absence of p53 or p16/p19 tumor

suppressor proteins. At difference to the generally reported protumoral role of

IKKβ in epithelial carcinogenesis, IKKβ showed a strong tumor suppressor

activity in skin tumorigenesis that is independent of p53; interestingly, it is




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mediated by the proteins coded by the Ink4a/Arf locus (p16 and/or p19). In the

absence of p16 and p19, we found that IKKβ promotes the emergence of

spindle SCCs, a rare variant of malignant skin tumor. These results have

implications for the implementation of antitumoral or anti-inflammatory therapies

in skin, as IKKβ is considered a potential target for the development of these

types of interventions.




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Materials and Methods

Mice and treatments

Mouse experimental procedures were performed according to European and

Spanish regulations and were approved by the Ethics Committee for Animal

Welfare of CIEMAT and by the legal authority (protocol codes BME02/10 and

PROEX086/15). Transgenic mice used in this work have been described

previously: line L1of K5-IKKβ mice in B6D2 hybrid background (20); TgAC

(FVB/NTac-Tg(Hba-x-v-Ha-ras)TG.ACLed) mice (21), p53EKO mice (22,23), and

Ink4a/Arf KO mice (lacking p16 and p19 proteins in FVB background (24)). K5-

IKKβ mice are available at European Mouse Mutant Archive (code EM: 09179).

The mice used in this study were viable and lack any evident alteration. In the

breedings, all the genotypes were obtained in the expected ratios.

For two-stage skin carcinogenesis experiments, back skins of mice were

initiated two days after shaving by treatment with 100 μg of 7,12-

Dimethylbenz[a]anthracene (DMBA, Sigma Aldrich), and promoted with 5 μg of

12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma Aldrich) in 200 μl of

acetone twice weekly during 12 weeks. In carcinogenesis experiments in TgAC

background, skins were TPA-treated twice weekly for 12 weeks. Tumor number

and size were recorded weekly. For bromodeoxyuridin (BrdU) labelling, mice

were injected intraperitoneally with BrdU (0.1mg/g weight, Sigma-Aldrich); 1.5 h

later, mice were sacrificed and the skin was harvested. For hyperplasia

induction mice back skin was shaved and topically treated twice with 5 μg of

TPA or vehicle at days 3 and 5 after shaving.

Genotyping of mice




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Genotyping of K5-IKKβ and of p53 and Ink4a/Arf loci were performed by PCR

analysis of tail DNA, as previously described [(20), (23), (25)].

Histology and immunohistochemistry

Mouse tissues were dissected and fixed in 10% buffered formalin or 70%

ethanol and embedded in paraffin. Five μm-thick sections were used for H&E

staining or immunohistochemical preparations. Antibodies references are in

Supplementary Material and Methods. Immunoreactivity was revealed using an

ABC avidin-biotin-peroxidase system and ABC substrate (Vector Laboratories),

and counterstained slightly with haematoxylin.

Quantification of BrdU staining and vessel density

For quantification of BrdU staining in Fig. 1E, the number of BrdU-positive cells

in 600 to 800 basal cells in 4 epidermal hyperplasias from 4 TgAC/K5-IKKβ

mice and in 6 papillomas from 4 TgAC mice were counted. For vessel density

(Fig. 1F), the number of blood vessels were counted in 10 different fields (20x

objective) in 5 different TgAC mice and 4 different TgAC/K5-IKKβ mice topically

treated with TPA for 3 weeks.

Determination of epidermal CD34+ cells

For the flow cytometry analysis shown in Supplementary Fig 2B-C, mean values

were obtained from the analysis of five individual K5-IKKβ and wt mice. Similar

results were obtained in three independent experiments. The images shown in

Supplementary Fig 2D-E are representative of the data obtained in the analysis

of 3 wt and 3 K5-IKKβ 10-week-old mice. Data in Supplementary Fig 2F-H

comes from the analysis of 4 animals of each genotype.

Protein extraction and western blot analysis




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Whole-cell protein extracts from mouse tumors and tissues were prepared using

standard techniques. Protein content was determined by the Bradford

colorimetric protein assay (BioRad Laboratories). Antibodies references are in

Supplementary Material and Methods.

Keratinocyte culture

Primary keratinocytes were obtained from skins of 2-day-old mice and cultured

as described previously (20). Plates with 3-day-cultured keratinocytes were

scrapped-off, pelleted and the protein extracts subjected to western blotting.

Statistical analysis

Data are expressed as mean ± SD. Statistical significance of data was

assessed using the unpaired, two-tailed Student´s t-test or Fisher’s exact test. P

values < 0.05 were considered significant.




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Results

IKKβ activity protects against skin cancer

With the aim of determining the role of IKKβ in non-melanoma skin cancer, we

performed DMBA-TPA carcinogenesis assays in transgenic mice

overexpressing IKKβ in basal cells of skin (K5-IKKβ mice) and in non-transgenic

littermates (n=17 for each genotype). K5-IKKβ mice developed fewer tumors

than wt mice (Fig. 1A); in addition, fewer K5-IKKβ mice generated tumors, and

those tumors were smaller than wt tumors (not shown); all these data suggest

an antitumoral activity of IKKβ in skin. Given the low tumor number obtained in

this carcinogenesis protocol, we aimed to confirm these data in other genetic

background more prone to skin tumorigenesis. We used TgAC mice, which

express a mutated Ha-Ras oncogene in skin (26). We generated cohorts of 9

mice bearing and 11 mice without the K5-IKKβ transgene in TgAC background,

which were subjected to topical treatment with TPA in order to induce papilloma

formation. Although 100% of the control TgAC mice developed papillomas by

week 7 of treatment, none of the TgAC/K5-IKKβ mice developed tumors at this

time point (p<0.0001), and only around half of them developed tumors by week

12 (not shown). Furthermore, the number of papillomas per mouse and their

size was markedly diminished in TgAC/K5-IKKβ mice compared to TgAC mice

(Fig. 1B-C). There were marked macroscopic and microscopic differences

between both genotypes: while Tg.AC lesions were truly exophytic papillomas

typical of this carcinogenesis protocol, the lesions obtained in TgAC/K5-IKKβ

were much smaller papillomatous epidermal hyperplasias (Fig. 1C), which

proliferated less and induced a weaker angiogenic response than TgAC




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papillomas (Fig. 1D-E). This weaker angiogenic response was also observed in

TgAC/K5-IKKβ skin TPA-treated for 3 weeks (before the development of skin

lesions) when compared to TgAC skin (Fig. 1F). These results indicate that

IKKβ precludes skin tumor formation. Transgenic mice express IKKβ in a variety

of tissues; the marked difference in IKKβ effect over tumorigenesis between

skin and oral epithelia do not seem to be related with differences in the

expression level of IKKβ between both tissues (19).

K5-IKKβ skin shows minor differences in immune cell populations

Inflammation by infiltrating immune cells facilitates the acquisition of the

fundamental features of cancer (27) and deeply affects skin tumor development

(28). One important function of IKKβ is the regulation of the inflammatory

process by controlling NF-κB activity. So, the antitumoral activity of IKKβ in skin

could be mediated by differences in skin resident immune cell populations. We

studied by flow cytometry the number of different immune cells both in

epidermis and in dermis of wt mice and K5-IKKβ littermates (Supplementary

Fig. S1A-B). Interestingly, the percentage of CD45+ cells is roughly double in

dermis than in epidermis of normal mice (9.3% vs. 4.3%); these cells are mainly

T lymphocytes (CD3+ cells), both inflammatory (CD3+, Tβ+) and skin-resident T

cells (CD3+, Tγδ+). We also detected other immune cell populations potentially

relevant in carcinogenesis, as macrophages (CD11b+); dendritic cells (CD11c+);

and natural killer cells (Supplementary Fig. S1A). In K5-IKKβ epidermis, we

observed a modest, non-significant decrease in CD3+ cells (both in Tβ+ and in

Tγδ+ subpopulations), a slight increase in macrophages and a more prominent

diminution in NK cells (Supplementary Fig. S1B). In dermis, all the populations




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studied were roughly equally abundant in Tg and in wt mice. From these results,

we conclude that the minor changes observed in the frequency of immune cells

(T-lymphocytes and myeloid cells) do not seem responsible for the marked

differences in skin cancer susceptibility between K5-IKKβ Tg and wt mice.

Characterization of the stem cell population in skin of K5-IKKβ transgenic

mice

The decreased susceptibility to tumoral transformation in K5-IKKβ mice could

be related to differences in the amount of cells able to originate a tumor. CD34

is a cell surface marker expressed by skin stem cells in the bulge region of the

hair follicles (29), which is needed for papilloma formation in mice (30). CD34+

cells become more abundant during the process of malignization (31), and

CD34+ cells isolated from primary tumors can originate new tumors if

transplanted to secondary recipients (32). Therefore, CD34 is considered a

marker of stem cells and of cancer stem cells in skin. In order to assess if the

lower frequency of skin cancer in IKKβ overexpressing mice is related to a

reduction in the number of skin CD34+ cells, we studied the abundance and cell

cycle distribution of these cells (Supplementary Fig. S2A-C). Surprisingly, K5-

IKKβ mice have roughly double the number of CD34+ cells than their non-

transgenic counterparts (Supplementary Fig. S2A-B). As expected, these

CD34+ cells were located in the hair follicle bulge both in wt mice and in K5-

IKKβ mice skin (Supplementary Fig. S2D-E), but they seem to cycle slightly less

actively in Tg than in wt mice (statistically non-significant, Supplementary Fig.

S2C). In BrdU label-retaining assays, we did not detect a reduction in the stem

cell population in K5-IKKβ mice (Supplementary Fig. S2F-H). Therefore, we




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conclude that the decreased sensitivity to skin cancer found in K5-IKKβ mice is

not due to the lack of stem cells able to generate tumors, or to differences in

their location or proliferative state.

K5-IKKβ keratinocytes express increased levels of tumor suppressor

genes.

In order to understand the differences observed in tumoral predisposition

between wt and K5-IKKβ transgenic skin, we studied the expression of several

TSGs important in tumoral transformation. We analyzed p53 (encoded by the

TSG most frequently mutated in cancer), p19 and p16 proteins (products of the

transcription of the Ink4a/Arf locus, implicated in the functionality of p53 and Rb

proteins, respectively), and p21 (a protein transcriptionally regulated by p53 that

controls cell cycle progression). As the analysis of these proteins in complete

skin by western blot is not appropriate, due to the variety of cellular types

present in skin, we studied cultured skin keratinocytes from K5-IKKβ and wt

mice. We consistently found that transgenic keratinocytes express increased

amounts of p53, p16, and p19 (Fig. 2A). We confirmed the result for p19 by

immunohistochemical staining in TPA-treated skin sections (Fig. 2B-C: 62% of

basal nuclei were intensely stained in Tg skin, at difference with the weak

staining found in 24% of the nuclei in wt mice). In accordance with the data

obtained in the western blot, we found, in RT-qPCR analysis of RNAs from Tg

and wt cultured skin keratinocytes, a significant increase for p16, p19 and p53

in Tg keratinocytes, but not for p21 (Fig. 2D). Altogether, these results suggest

that the skin tumor-protective activity of IKKβ could well be mediated by the

increased amount of p16, p19 and p53 in skin keratinocytes.




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The skin tumor suppressive activity of IKKβ is independent of p53

We aimed to analyze the implication of p53 in the skin tumor suppressive

activity of IKKβ. To this end, we crossed K5-IKKβ mice with p53fl/fl/K14-Cre mice

(22,33)]; these mice efficiently delete p53 exons 2 to 10 in the vast majority of

epidermal cells (23), and so we named them p53EKO mice; mice carrying both

genetic modifications (i.e., p53EKO/K5-IKKβ mice), simultaneously lack p53 and

overexpress IKKβ in skin basal proliferative cells, where skin tumors arise. We

performed a two-stage skin carcinogenesis experiment and compared the rate

of tumor appearance, tumor size, and histological classification between

p53EKO/K5-IKKβ and control p53EKO mice (Fig. 3). Interestingly, p53EKO/K5-IKKβ

mice also showed a great decrease in the mean number of skin tumors per

mice along all the experiment (Fig. 3A), similar to the results obtained in p53wt

background (Fig. 1A-B). Also, skin tumors in p53EKO/K5-IKKβ mice were smaller

than in p53EKO mice, being the proportion of medium and large tumors (diameter

of 2 to 5 mm, and >5 mm, respectively) greater in p53EKO mice (Fig. 3B).

Macroscopically, most tumors arising in p53EKO/K5-IKKβ mice were small,

exophytic and papillomatous (although four of the animals of this genotype

developed only one tumor each of faster growth and apparently more

malignant, with ulceration and infiltration of the subcutaneous tissue, not

shown). Most p53EKO mice, in contrast, developed multiple sessile infiltrating

tumors, presumably malignant. We assessed histologically 51 p53EKO and 26

p53EKO/K5-IKKβ tumors collected at weeks 13-18 (Fig. 3C). We found several

tumor types, and classified them as benign tumors (including squamous

papillomas, trichoepitheliomas, adenosquamous tumors and basosquamous

tumors) or malignant tumors (including squamous papillomas with invasive




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microcarcinoma foci and more malignant undifferentiated SCCs). Interestingly,

the percentage of malignant tumors was lower in p53EKO/K5-IKKβ mice than in

p53EKO mice (Fig. 3C). Representative examples of these tumors are shown in

Fig. 3D-G. Benign tumors keep a clear basal membrane that prevents invasion

(arrowheads in Fig. 3D-E). Carcinomas were frequently accompanied by

inflammatory cells, and showed highly dysplastic keratinocytes invading

underlying dermis, with condensed nuclei (karyopyknosis), variable in size

(anisokariosis), probably as a consequence of p53 absence (asterisks in Fig.

3F-G).The tendency to develop of less malignant tumors in p53EKO/K5-IKKβ

mice was further confirmed by the decreased expression of markers associated

to malignant transformation, such as keratin K13 and P-Akt, even in the few

SCCs obtained in this genotype (Supplementary Fig. S3). In summary, these

results indicate that IKKβ overexpression leads to the development of fewer and

less malignant tumors in mice lacking skin p53. Western blot analysis of SCCs

from both genotypes confirmed that tumors originated from cells lacking p53, as

expected (Fig. 3H). In addition, p53EKO/K5-IKKβ carcinomas showed increased

p19 and p16 levels, in accordance with the results shown in Fig. 2A for cultured

keratinocytes, and also of p21. When we studied Stat-3 and Akt, two

proliferative pathways frequently activated in carcinogenesis, we did not find

marked differences between p53EKO and p53EKO/K5-IKKβ tumors. Altogether,

these results indicate that the tumor suppressive activity of IKKβ in skin is not

mediated by p53.

In addition to the DMBA/TPA carcinogenesis protocol, we also studied the

combined effect of IKKβ overexpression and p53 absence over spontaneous

tumorigenesis. We generated additional cohorts of p53EKO and p53EKO/K5-IKKβ




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mice, followed tumor appearance over time, and classified histologically the

tumors generated (Table 1A). Skin tumors were mainly SSCs of different

degrees of differentiation. Interestingly, the percentage of mice with tumors was

lower in p53EKO/K5-IKKβ than in p53EKO mice, and the mean number of tumors

per mice with tumors was also lower in p53EKO/K5-IKKβ mice (1.4 vs. 1.8).

Furthermore, the percentage of skin tumors was roughly half in mice

overexpressing IKKβ (38% vs 78%; p<0.001). Collectively, these results

reinforce, in a different experimental in vivo situation, the protective effect of

IKKβ in skin cancer and confirm its independence from p53.

Ink4a/Arf locus mediates the skin tumor suppressive activity of IKKβ

We next wondered if the tumor-suppressive activity of IKKβ could be mediated

by p16 and p19, which are coded by the same Ink4a/Arf locus. Heterozygous

mice for an Ink4a/Arf KO allele (24) were crossed with K5-IKKβ mice and

backcrossed with FVB mice for three generations; we obtained mice

homozygous for the Ink4a/Arf KO allele (lacking p16 and p19 in all their cells),

and simultaneously bearing the K5-IKKβ transgene. We performed a

DMBA/TPA skin carcinogenesis experiment in 11 Ink4a/Arf KO mice and 12

Ink4a/Arf KO/K5-IKKβ mice. This experiment could not be extended for more

than 14 weeks after DMBA application due to humanitarian reasons, given the

fast growth or the malignant aspect of some of the tumors. Interestingly, in the

absence of the proteins coded by the locus Ink4a/Arf, there were no differences

in tumor burden between animals carrying or lacking the K5-IKKβ transgene

(Fig. 4A). The histopathological classification of the tumors analyzed (121 in

total) is shown in Fig. 4B. Of note, due to the absence of p16 and p19, and in




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spite of the relatively short period of time of the experiment, the majority of the

tumors were SCCs of varying levels of differentiation. By contrast, K5-IKKβ

mice bearing two wt copies of the Ink4a/Arf locus resulted in a marked lower

amount of SCCs that arose at later times than SCCs in Ink4a/Arf KO

background. In addition, all the SCCs observed in Ink4a/Arf wt/wt were well

differentiated SCCs (Supplementary Fig. 4A-C).

Interestingly, in contrast to the results obtained in wt and p53EKO backgrounds

(Figs. 1 and 3), malignancy was augmented in Ink4a/Arf KO/K5-IKKβ tumors, as

the percentage of undifferentiated SCCs and spindle cell SCCs (SpSCCs) was

much higher in Ink4a/Arf KO/K5-IKKβ mice than in Ink4a/Arf KO mice (Fig. 4B;

p<0,0001). Representative examples of SCCs with increased levels of

malignancy, from well-differentiated SCCs to highly undifferentiated cutaneous

SpSCCs are shown in Fig. 4C-H. SpSCC is a rare variant of SCC in which

tumor cells with spindle-shaped appearance infiltrate the dermis diffusely,

without formation of epithelial nests or cords (34); this tumor type is so

malignant and undifferentiated that determination of its origin is sometimes

doubtful since spindle keratinocytes appearance is very similar to that of

malignant cells from fibrosarcomas or melanomas. We have studied the

expression patterns of keratin K5, vimentin and protein S100 (epithelial,

mesenchymal and melanocytic cell markers, respectively), in order to ensure

the epidermal origin of uncertain tumors containing spindle cells and in some

other tumors with clear classification; we analyzed 19 tumors from 9

Ink4a/ArfKO/K5-IKKβ mice and 8 tumors from 4 Ink4a/Arf KO mice (Table 2).

Typically, SpSCCs co-express K5 and vimentin (Fig. 4I-J) with the absence of

S100 (Fig. 4K; compare with the staining in a positive control shown in




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Supplementary Fig. S5A-B), which differentiate them from fibrosarcomas

(positive for vimentin but negative for keratins) and spindle melanomas, tumors

that have been described in Ink4a/Arf KO mice. The low percentage of

fibrosarcomas in comparison with the data published in (24) could be due to

differences in genetic background, latency time or tumorigenesis treatment

(DMBA/UV in (24) and DMBA/TPA in this work).

Of note is that we have been able to determine the follicular origin of SpSCCs,

as K5-positive epidermal follicular cells infiltrate the underlying dermis, showing

highly dysplastic spindle shape cells and giving rise to a SpSCC

(Supplementary Fig. S5C). We have not found consistent differences in the

activity of Stat-3, Erk or Akt pathways between tumor histological types or

genotypes (Supplementary Fig. S5D). Spontaneous tumorigenesis affected

slightly more strongly to Ink4a/Arf KO/K5-IKKβ than to control Ink4a/Arf KO mice,

as a higher percentage of mice developed tumors (Table 1B). In addition,

although the number of tumors is low, a tendency to develop more malignant

skin tumors was also observed (as 2 out of 6 skin tumors in Ink4a/Arf KO/K5-

IKKβ were highly malignant SpSCCs, while this tumor type was not observed in

Ink4a/Arf KO mice), reinforcing the data obtained in two-stage skin

carcinogenesis experiments.

Taken together, these results indicate that the antitumoral effect of IKKβ is

mediated by p16 or p19; even more, in the absence of these tumor

suppressors, IKKβ is not able to protect against skin tumor appearance, but

instead it seems to increase tumor malignancy, as described for other tissues.




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Discussion

IKKβ is a fundamental multitarget kinase (5), and mice lacking IKKβ in all their

cells die during gestation due to excessive hepatic apoptosis (35,36); the

absence of IKKβ in epidermis and stratified epithelia results in death at the

beginning of postnatal life by an inflammatory skin disease mediated by tumor

necrosis factor (37). In humans, however, IKKβ deletion seems to be less

harmful, as homozygous deletion of the IKBKB gene is not lethal, at least in

some cases, but leads to immunodeficiency (38). In agreement with the multiple

functions of IKKβ, its effect on tumoral transformation of the cells can be

mediated by different pathways, such as NF-κB activation (39); adaptation to

metabolic and oxidative stresses, increasing the ability of cancer cells to survive

at low glutamine concentration (40); establishment of stemness properties by

Wnt pathway regulation (41); modulation of T cell-dependent antitumoral

immune response (42); or inactivation of important cell cycle controllers as p53

(2) and p16 (3) among others. As a result of these pleiotropic functions, IKKβ

promotes cellular transformation and tumor growth in lung, pancreas, and oral

epithelia, among others (16,17,19), but it also results in an antitumoral effect in

myeloid cells during melanoma tumorigenesis (18). There are two recent

reports that illustrate clearly this context-dependent pro- or anti-tumoral dual

activity of IKKβ, as they report that IKKβ in mesenchymal cells has both tumor

promotor and tumor suppressor roles in intestinal tumorigenesis, probably as a

consequence of subtle differences in the subpopulations targeted in these

experiments (43,44). Here, we have found a surprising antitumoral role of

epidermal IKKβ in non-melanoma skin cancer (NMSC). This activity does not

seem to be exerted by modifications in the epidermal or dermal inflammatory




20

milieus, nor by a diminution in the amount of stem cells. As skin-specific p65

knock out leads to downregulation of NF-κB and also protects against tumoral

transformation (8), the effect of IKKβ overexpression over skin cancer is

probably mediated mainly by NF-κB-independent pathways. When we searched

for possible mediators of this skin tumor-protective function of IKKβ, we found

that K5-IKKβ epidermal keratinocytes expressed high levels of several tumor

suppressor proteins (p53, p16 and p19) that could explain the reduced

sensitivity to tumoral transformation of IKKβ-overexpressing skin.

Experimental skin carcinogenesis in animals lacking epidermal p53 indicated

that the antitumoral activity of IKKβ in skin is independent of p53; so, probably

the increased p53 protein level found in K5-IKKβ keratinocytes is secondary

and not directly caused by IKKβ. Interestingly, the antitumoral effect of IKKβ is

even stronger in a p53EKO background than in a p53wt background (Figs. 3A and

1A); this could be explained by the mutual inhibitory activity described for IKKβ

and p53, that would weaken the effect of IKKβ in the presence of p53 (2,45).

IKKβ protection against skin cancer, on the contrary, acts through p16, p19, or

both. Although some of the activities of p16 and p19 proteins are different, and

even opposite (46), both of them have strong and non-redundant tumor

suppressor activity. The lack of either p16 or p19 alone results in increased

cancer susceptibility, and is less harmful than the combined absence of both

proteins (47). Favoring the possible leading role of p19 over p16 as mediator of

skin IKKβ antitumoral activity are the facts that p19 is expressed at higher level

than p16 in skin keratinocytes in RT-qPCR experiments and that mice with one

of these proteins knocked-out confirm the higher general importance of p19

(47). If the main actor in IKKβ-mediated tumor protection were p19, it would




21

exert its antitumoral function by p53-independent functions, as p53 absence

does not diminish the skin antitumor activity of IKKβ. In any case, the

generation of K5-IKKβ mice lacking p16 or p19 individually and the study of

their sensitivity to skin cancer would be required to clarify which of these

proteins mediates IKKβ skin suppressive cancer functions.

It is interesting to highlight that skin overexpressing IKKβ gives rise, in the

absence of p16 and p19, to SpSCCs, both after DMBA/TPA treatment and

spontaneously (Fig. 4B and Table 1B). Spindle cell SCC is a rare type of skin

tumor, more frequent in immunosuppressed individuals. It has been described,

in two-stage chemical skin carcinogenesis, that SpSCC is associated to lower

expression level or to deletion of p16 and p19 (28). Our results also suggest

that this malignant carcinoma is favored by increased expression of IKKβ, as it

is found preferentially in Ink4a/Arf KO/K5-IKKβ.

The generation in recent years of a growing body of expression and genomic

data from tumoral samples supports the idea that IKKβ is a truly important

molecule in human cancer development. So, the data included in the catalogue

of somatic mutations in cancer (cosmic; http://cancer.sanger.ac.uk/cosmic)

indicate that IKKβ is frequently overexpressed (sometimes in association with

copy number gain) in several human cancers, mainly in esophagus (24.8% of

tumors), large intestine (18.5%) and breast (9.6%). Interestingly, IKKβ is also

overexpressed in more than 7% of the samples of malignant melanomas and of

stomach, ovary and lung cancers; unfortunately, there are not data available for

NMSC. Data in The Cancer Genome Atlas (TCGA) indicate that IKKβ is altered

(mostly amplified) in around 5% of head and neck squamous carcinomas; in 5-

10% of the cases of ovarian serous cystadenocarcinoma, colorectal and




22

stomach adenocarcinomas, liver hepatocellular adenocarcinoma, uterine corpus

endometrial carcinoma, lung adenocarcinoma, lung SCCs, and esophageal

carcinoma; and over 10% of the cases in breast invasive carcinoma, and

bladder urothelial carcinoma. Interestingly, TCGA data indicates that IKKβ is not

altered in NMSC, although the number of samples analyzed is low (48,49).

Overall, these data confirm and expand the growing body of evidence obtained

in genetically modified animal models, indicating that IKKβ overexpression

favors tumor growth in many cells types, but not in epidermal keratinocytes,

where IKKβ prevents tumor development. This represents a warning note

against the proposed treatment of inflammatory and tumoral diseases with IKKβ

inhibitors (50), as ubiquitous pharmacological IKKβ inhibition could favor skin

tumor appearance.

In summary, we have found and described for the first time a strong skin tumor

suppressive function of IKKβ. Mechanistically, this antitumoral activity is

mediated by p16 or p19, but not by p53. In addition, IKKβ seems to cooperate

with lack of INK4A/ARF in the genesis of SpSCCs. Overall, these results draw

attention to the need of a careful evaluation of therapies aimed to IKKβ

inhibition in the treatment of inflammatory and tumoral diseases.




23

Acknowledgements

This research was supported partially by funds from Fondo Europeo de

Desarrollo Regional (FEDER). We would like to thank Rebeca Sanz, Berta

Hernanz, Nerea Guijarro for their valuable technical help; Rebeca Sánchez-

Domínguez and Omaira Alberquilla for their help with the flow cytometry

studies; Federico Sánchez-Sierra and Pilar Hernández for their excellent

histological processing of the samples; and Edilia de Almeida and the personnel

of the CIEMAT Animal Unit for mice care and for their help with mice

treatments. We also thank Manuel Serrano (Centro Nacional de Investigaciones

Oncológicas, CNIO, Spain) for his generous gift of Ink4a/Arf KO mice. Thanks to

Anton Berns (Netherlands Cancer Institute, NKI, The Netherlands) for supplying

with p53EKO mice. The results shown here are in part based upon data

generated by the TCGA Research Network (http://cancergenome.nih.gov/).




24

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28

Tables

Table 1: Histopathological classification of spontaneous tumors in: A) p53EKO

and p53EKO/ K5-IKKβ mice; and B) Ink4a/Arf KO and Ink4a/Arf KO/K5-IKKβ mice.

A B

p53EKO p53EKO/K5-IKKβ Ink4a/Arf KO Ink4a/Arf KO/K5-IKKβ

Mice analyzed 44 45 28 40

Mice with tumors (%) 23 (52%) 17 (38%) 5 (18%) 18 (45%)

Total tumor number 41 24 7 23

Tumors per mouse 1,8 1,4 1,4 1,3

Skin tumors (*) 32 (78%) 9 (38%) 2 (29%) 6 (26%)

Other tumors (*, **) 9 (22%) 15 (62%) 5 (71%) 17 (74%)

Average age of skin tumor appearance (months)

10 9,5 8,5 6,5

(*) The percentage versus total tumor number is shown.

(**) Non-skin tumors obtained in p53EKO background were 4 oral and 5

mammary gland tumors in p53EKO mice, and 9 oral and 6 mammary gland

tumors in p53EKO/K5-IKKβ mice.

(**) Non-skin tumors obtained in Ink4a/Arf KO background were 5 hematological

tumors in Ink4a/Arf KO mice, and 4 hematological and 13 oral tumors in

Ink4a/Arf KO/K5-IKKβ mice.




29

Table 2: Immunohistochemical classification of uncertain SCCs and

fibrosarcomas arisen in Ink4a/Arf KO/K5-IKKβ and Ink4a/Arf KO mice treated with

DMBA/TPA. Classification is based on staining for keratin K5, vimentin and

S100, as described in the text. Some well-differentiated and undifferentiated

SCCs were included for control purposes.

Genotype Tumor type Total Well-diff. Undifferentiated Spindle cell Fibrosarcomas

SCCs SCCs SCCs Ink4a/Arf KO 0 5 14 0 19

/K5-IKKβ Ink4a/Arf KO 3 3 1 1 8




30

Figure Legends

Figure 1. Protective function of IKKβ in mouse skin tumorigenesis. A) Tumor

multiplicity in K5-IKKβ transgenic mice and wt littermates in C57BL6/J x DBA2/J

hybrid background. The low number of tumors per mouse is due to the low

sensitivity to skin carcinogenesis protocols of the mice in this background. B)

Tumor multiplicity in K5-IKKβ transgenic mice and control littermates in TgAC

background. Tumor burden is much more elevated than in Fig. 1A by the

increased sensitivity of TgAC mice to tumor development. C) Examples of the

macroscopic appearance of TgAC and TgAC/K5-IKKβ mice subjected to TPA

treatment at week 12 and examples of the H&E staining of one of the lesions

obtained. TgAC mice developed numerous exophytic papillomas generally

greater than 4 mm in diameter. In contrast TgAC/K5-IKKβ developed a lower

number of smaller tumoral lesions. Note that the TgAC/K5-IKKβ mouse shown

in the macroscopic image is the one with the highest number of tumoral lesions

in this genotype. D) Representative images of the immunohistochemical

analysis of IKKβ expression, proliferation (measured as BrdU incorporation) and

blood vessels supply (Sma expression), in tumoral lesions of the indicated

genotypes. E) Quantification of BrdU staining. F) Quantification of the number

of blood vessels in the dermis of TPA-treated skins of the indicated genotypes.

* indicates p<0.05; ** indicates p<0.01. Scale bars: 100 μm.

Figure 2. Increased expression of p53, p19 and p16 in K5-IKKβ epidermal

keratinocytes. A) Western blot analysis of cultured skin keratinocytes. B-C)

Immunohistochemical staining of p19 in hyperplastic back skin sections of the

indicated genotypes; note the increased staining of basal skin cells of K5-IKKβ




31

transgenic mice compared to wild type mice (arrowheads). D) RT-qPCR

analysis of the expression of several tumor suppressor proteins in RNA

samples from skin keratinocytes of transgenic K5-IKKβ and wild-type mice.

Figure 3. p53 is not needed for the IKKβ tumor protective activity. A) Tumor

multiplicity in K5-IKKβ transgenic mice and control littermates in p53EKO

background subjected to a two-stage DMBA/TPA carcinogenesis protocol (n=7

in each group). Differences between both genotypes from week 8 to week 15

are statistically significant (p<0.01; Student’s t-test). B) Size distribution of skin

tumors along the experiment. Note the reduced percentage of large- and

medium-sized tumors in p53EKO/ K5-IKKβ mice in comparison to p53EKO mice.

C) Histopathological classification of tumors obtained in p53EKO and p53EKO/K5-

IKKβ mice after treatment with DMBA and TPA. The percentages of malignant

tumors are indicated (p<0.001, Fisher’s exact test). D-G) Histological aspect of

benign and malignant tumors of the indicated genotypes; D,E are benign

squamous papillomas and F,G undifferentiated SCCs. H) Western blot analysis

of important proteins for tumor progression in 3 SCCs arisen in mice of each of

the indicated genotypes. In the lane marked sk, an extract from K5-IKKβ

cultured skin keratinocytes was loaded. Note increased p21, p19 and p16 levels

in p53EKO/K5-IKKβ carcinomas. Scale bars: 100 μm.

Figure 4. Deletion of the Ink4a/Arf locus abrogates the tumor protective activity

of IKKβ. A) Tumor multiplicity in K5-IKKβ transgenic mice and control littermates

in Ink4a/Arf KO background subjected to a two-stage DMBA/TPA carcinogenesis

protocol. B) Histopathological classification of tumors obtained in Ink4a/Arf KO




32

and Ink4a/Arf KO/K5-IKKβ mice 14 weeks after the beginning of the treatment

with DMBA and TPA. C–H) Histological aspect of skin tumors with increased

levels of malignancy in the indicated genotypes: (C, F), well differentiated

SCCs, showing abundant foci of squamous differentiation (horny pearls,

arrows); (D, G), moderately differentiated SCCs, with nests and cords of

keratinocytes infiltrating the underlying dermis and muscle; (E, H): highly

undifferentiated SpSCCs showing spindle shape keratinocytes infiltrating the

dermis in a diffuse pattern. I-K) Immunohistochemical staining of a SpSCC for

keratin K5 (I), vimentin (J) and S100 (K). Scale bars: 100 μm.




Figure 1

A

B

C

E

0

10

20

30

40

50

TgAC TgAC/ K5-IKKβ

% o

f B

rdU

+ c

ells

F

0

40

80

120

160

200

240

TgAC TgAC/ K5-IKKβ

blo

od v

essels

/10 fie

lds

TgAC TgAC/K5-IKKβ

D TgAC TgAC/K5-IKKβ

IKKβ IKKβ

BrdU BrdU

Sma Sma

0

1

2

3

4

5 6 7 8 9 10 11 12 13 14 15 16 17 18

tum

ors

/ m

ouse

weeks of treatment

wt K5-IKKβ

0

20

40

60

80

5 6 7 8 9 10 11 12 13 14 15 16 17 18

tum

ors

/ m

ouse

weeks of treatment

TgAC TgAC/K5-IKKβ




Figure 2

D

0

1

2

3

4

5

6

7

IKKβ p53 p21 p16 p19

rela

tive e

xp

ressio

n

wt K5-IKKβ

A

IKKβ

p19

p53

p16

Actin

wt K5-

IKKβ

p21

B

C

wt

K5-IKKβ




0% 50% 100%

p53EKO/ …

p53EKO

Benign tumors Malignant tumors

p53EKO

p53EKO/

K5-IKKβ (n = 26)

(n = 51)

C

39%

19%

Figure 3

B

tum

ors

/ m

ouse

p53EKO/K5-IKKβ

E

F

*

G

*

D

p53EKO

p53EKO p53EKO/

K5-IKKβ

IKKβ

P-Stat3

P-Akt

p53

p21

p19

p16

Actin

1 2 3 4 5 6 sk

H

Stat3

Akt

K5-

IKKβ

0.0 0.0 0.1 2.1 1.8 2.0

0.9 1.2 1.2 1.0 1.2 0.7

1.1 1.4 1.5 1.4 0.7 1.1

0.4 0.2 0.4 0.2 0.2 0.2

0.5 0.8 1.4 1.7 1.4 1.6

0.8 1.0 0.8 2.7 1.9 1.2

1.2 0.7 1.5 1.7 1.4 1.2

1.0 1.1 1.2 0.7 1.0 0.7

1.3 1.3 1.3 1.2 0.9 0.9

0

2

4

6

6 8 10 12 14

weeks of treatment

0

10

20

30

40

6 8 10 12 14

< 2 mm 2-5mm >5mm

A

0

10

20

30

40

6 8 10 12 14tum

ors

/ m

ouse

weeks after treatment

p53EKO

p53EKO/K5-IKKβ

p53EKO

p53EKO/K5-IKKβ

p53EKO

p53EKO/K5-IKKβ




21%

14%

32%

27%

3% 3%

Ink4a/Arf KO

(n = 63)

Benign tumors

Tumors with microcarcinomas

Well differentiated SCCs

0

4

8

12

7 8 9 10 11 12 13 14

tum

ors

/ m

ou

se

weeks after treatment

Ink4aKO

Ink4aKO/K5-IKKβ

Figure 4

A Ink4a/Arf KO

Ink4a/Arf KO/K5-IKKβ

31%

0 19%

12%

14%

24%

Ink4a/Arf KO/ K5-IKKβ (n = 58)

Moderately differentiated SCCs

Undifferentiated SCCs

Spindle cell SCCs

B

K5 Vimentin S100

D

F

E

Ink4a/A

rf K

O

C

G

K J I

H

Ink4a/A

rf K

O/ K

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In

k4a/A

rf K

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5-I

KKβ




Published OnlineFirst June 5, 2017.Mol Cancer Res Angustias Page, Ana Bravo, Cristian Suárez-Cabrera, et al. Ink4a/Arf-dependent

-mediated Resistance to Skin Cancer Development isβIKK

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