Klotho Structure-Function Relationships in Breast Cancer Tumor Suppressor

Author Manuscript Published OnlineFirst on June 25, 2015; DOI: 10.1158/1541-7786.MCR-15-0141 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Klotho structure-function relationships in breast cancer

Tumor Suppressor Activity of Klotho in Breast Cancer is Revealed by Structure-function Analysis

Hagai Ligumsky1, 2#, Tami Rubinek1, Keren Merenbakh-Lamin1, 2, Adva Yeheskel3, Rotem Sertchook4, Shiri Shahmoon1, 2, Sarit Aviel-Ronen5 and Ido Wolf1, 2

1 The Oncology Division, Tel Aviv Sourasky Medical Center, Israel; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 3Bioinformatics Unit, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel; 4Bioinformatics and Biologial Computing Unit, Weizmann Institute of Science, Rehovot, Israel. 5Department of Pathology, Chaim Sheba Medical Center, Ramat Gan, Israel.

Running title: Klotho structure-function relationships in breast cancer

To whom correspondence should be addressed: Ido Wolf, Oncology Division, Tel Aviv Sourasky Medical Center, 64239, Tel Aviv, Israel. Phone: 972-52-736-0558 FAX : 972-3- 697-3030. E-mail: [email protected]

# This work was performed in partial fulfillment of the requirements for a Ph.D. degree.

Key words: klotho, breast cancer, KL1, KL2, glucuronidase

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 25, 2015; DOI: 10.1158/1541-7786.MCR-15-0141 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Klotho structure-function relationships in breast cancer

Abstract

Klotho is a transmembrane protein containing two internal repeats, KL1 and

KL2, both display significant homology to members of the β-glycosidase family. Klotho is

expressed in the kidney, brain and various endocrine tissues, but can also be cleaved

and act as a circulating hormone. Klotho is an essential cofactor for binding of fibroblast

growth factor 23 (FGF23) to the FGF receptor and can also inhibit the insulin-like growth

factor-1 (IGF-1) pathway. Data from a wide array of malignancies indicate klotho as a

tumor suppressor; however, the structure-function relationships governing its tumor

suppressor activities have not been deciphered.

Here the tumor suppressor activities of the KL1 and KL2 domains were

examined. Overexpression of either klotho or KL1, but not of KL2, inhibited colony

formation by MCF-7 and MDA-MB-231 cells. Moreover, in vivo administration of KL1 was

not only well tolerated but significantly slowed tumor formation in nude mice. Further

studies indicated that KL1, but not KL2, interacted with the IGF-1R and inhibited the IGF-

1 pathway. Based on computerized structural modeling, klotho constructs were

generated in which critical amino acids have been mutated. Interestingly, the mutated

proteins retained their tumor suppressor activity but showed reduced ability to modulate

FGF23 signaling.

These data indicate differential activity of the klotho domains, KL1 and KL2, in

breast cancer, and reveal that the tumor suppressor activities of klotho can be dissected

from its physiologic activities.

Implications: These findings pave the way for a rational design of safe klotho-based

molecules for the treatment of breast cancer.

Klotho structure-function relationships in breast cancer

Introduction

Klotho (α-klotho) is a transmembrane protein expressed in the distal convoluted

tubules of the kidneys and the choroid plexus, as well as in various endocrine and

exocrine tissues (1), but it can also be cleaved, shed and act as a circulating hormone

(2, 3). Klotho-deficient mice manifest a syndrome resembling human aging, while

overexpression of klotho extends lifespan (1, 4). Major physiologic activities of klotho

include regulation of phosphate and calcium homeostasis as well as of metabolic activity

(5-7). To date, several mechanisms of action of klotho have been described. Klotho is an

essential co-factor for binding of fibroblast growth factor (FGF) 23 to the FGF receptor

(FGFRs) (5, 8), it modulates bFGF signaling (9, 10), inhibits the insulin and the insulin-

like growth factor (IGF)-1 pathways (4, 9) and regulates the activity of the transient

receptor potential vanilloid type 5 (TRPV5) calcium channel (11, 12). The common link

between these diverse activities has not been elucidated yet.

In the past years klotho has emerged as a potent tumor suppressor in a wide

array of malignancies including breast, pancreas, colon, gastric and lung cancers, as

well as in melanoma (9, 10, 13-16). We have previously shown that klotho is abundantly

expressed in normal breast and pancreatic tissues and that upon malignant

transformation its expression is reduced (9, 10, 17). Restoring klotho expression, or

treatment with the soluble protein, inhibited proliferation of breast and pancreatic cancer

cells (9, 10), and further studies indicated it as an inhibitor of the IGF-1 pathway (4, 9).

Mouse klotho is a 1014 amino-acid long protein (1). Its intracellular domain is

composed of ten amino acids and has no known functional role (1). The extracellular

domain is composed of a signal sequence (SS), responsible for directing the protein to

the cell surface, and two internal repeats, KL1 and KL2, each about 450-amino acids

long (1), which exhibit only weak similarity to each other (21% amino-acid identity) (1).

The discrete activities of each domain have not been well-characterized yet. Cleavage of 3

Klotho structure-function relationships in breast cancer

the extracellular domain of klotho can yield either the entire extracellular domain

(130kDa), or the discrete domains KL1 and KL2 (2, 18, 19). While only the full-length

protein can serve as a co-factor for the activity of FGF23 (5, 8), either the full-length

protein or KL1 can inhibit proliferation of pancreatic cancer cells (10).

Each klotho domain displays significant homology to members of the glycoside

hydrolase family 1 enzymes (1). Two glutamic acid residues are required for the

enzymatic activity of family 1 β-glycosidases, one acts as a nucleophile and the other as

an acid/base catalyst (20). While these critical amino acids are not conserved in klotho,

klotho was shown to hydrolyze β-glycoside and β-d-glucuronide (20). In support of these

activities, mutations of critical amino acids, considered to be essential for its enzymatic

activity, abolished the ability of klotho to increase TRPV5 currents (11). Yet, these

mutations did not affect its ability to inhibit Wnt signaling. To our knowledge, the role of

these residues in mediating klotho tumor suppressor activities has not been studied yet.

We aimed to decipher klotho structure-function relationships that are involved in

mediating its tumor suppressor activities in breast cancer. Our findings demonstrate that

KL1, but not KL2, can inhibit growth of breast cancer cells in vitro and in vivo. Structural

modeling and site-directed mutagenesis indicate that amino acids, essential for

modulation of FGF23 signaling, are not required for klotho tumor suppressor activity.

Materials and methods

Chemicals and antibodies

The chemicals used were FGF23, IGF-1 and soluble human KL1 (hKL1)

(PeproTech Inc, Rocky Hill, NJ), and G418 (Invitrogen). Antibodies used were phospho-

AKT1 (S473), phospho-IGF-IR (Y1131), total pan-AKT (Cell Signaling Technology),

diphosphorylated-Thr183/Tyr185 and total-ERK1/2 (Sigma) and HA (Covance). Anti-

Klotho structure-function relationships in breast cancer

klotho antibodies directed against KL1 domain (KM2076) and KL2 domain (KM2119)

were a kind gift from Kyowa Hakko Kogyo Co., Ltd.

Cells and transfections

MCF-7, MDA-MB-231, and HEK-293 cells were obtained from the American Type

Culture Collection (ATCC) (Manassas, VA).Cells were received from Dr. Hadassa

Degani's lab at Weizmann Institute of Science, which obtained them directly from the

ATCC and were not passaged more than 6 months after resuscitation. All transfections

used Lipofectamine 2000 (Invitrogen).

Structural model construction of KL1 and KL2 domains

Template structures for molecular modeling human klotho were selected by

BLAST search against the PDB. The BLAST search suggested the structures of the

human Cytosolic Neutral beta-Glycosylceramidase (Klotho-related Protein, KLrP) as best

template (42% sequence identity to KL1 domain and 33% to KL2 domain). A crystal

structure of KLrP with highest resolution (1.60 Å) and good stereochemistry was

selected (PDB entry 2E9L). Structural model of KL1 was built using PDB 2E9L (21) as a

template. The template was selected using HHPRED (22), The model was built using

Modeller (23). The model structure was refined using the Rosetta Relax protocol (v. 3.2)

(24). KL1 model was assessed using ModFold (25) and ConSurf (26), using a multiple

sequence alignment of 150 KL1 homologs collected from Uniprot (27) using Blast (28)

and aligned with Mafft (29). Structural model of KL2 was based on a sequence

alignment between KL2 domain, the template structure and 14 representative

sequences of family 1 Glycoside hydrolase, which was carried out using several multiple

alignment algorithms (Expresso, Promals3D and FFAS03). A set of 20 all-atoms model

of KL2 was built using the restrained-based modeling approach as implemented in the

program MODELLER 9V6 based on the different alignments. The model was evaluated 5

Klotho structure-function relationships in breast cancer

using MODELLER objective function, DOPE, ProSA and Anolea. The model showing the

best score as judged by these evaluation methods was saved for further studies and

presented here.

Generation of klotho expression constructs

Mouse HA-tagged full-length klotho (FL-KL) in pcDNA3.1 was a generous gift

from Y Nabeshima (Kyoto University, Japan). Mouse KL1 and KL2 constructs were

prepared in two steps, in order to contain the original FL-KL signal sequence (SS). For

the first step the SS was PCR amplified and restriction sites were introduced using

specific primers

5-ATCGAATTCATGCTAGCCCGCGCCCCTCCT-3 (EcoRI), and

5-ATCAAGCTTGGCGCGCAGGGCGAGCAGCAG-3 (HindIII). KL1 and KL2 were PCR

amplified and restriction sites were introduced using specific primers. For KL1 –5-

ATCAAGCTTATGCGCTGCCTGAGCGC-3 (HindIII) and

5-ATCCTCGAGTCTCTTCTTGGCTACAACCC-3 (Xho). For KL2 –

5-ATCAAGCTTAAACCTTACTGTGTTGATTTC-3 and

5-ATCCTCGAGTCTCTTCTTGGCTACAACCC-3 (Xho). Plasmids were digested with the

suitable enzymes and the SS was ligated to KL1 and KL2. Subsequently the constructs

were sequenced for verification and subcloned into pcDNA3.1 backbone.

Mouse KL1 with Intra-cytoplasmic/Trans membrane (IC/TM) was prepared as followed:

at first an IC/TM fragment was PCR amplified and restriction sites were introduced using

specific primers: 5'-ATCCTCGAGTCTTTGCTGGTCTTCATC-3' (XhoI)

and 5'-GATCTCGAGCTTATAACTTCTCTGGCC-3' (XhoI). Then the fragment and KL1

plasmid were digested with the suitable enzymes and the IC/TM was ligated to KL1.

Human FL-KL (hFL-KL) in pEF and human full-length without IC/TM (hFL-KL ΔIC/TM)

was a generous gift from M Kuro-O (UT Southwestern, USA).

Directed mutagenesis of klotho putative enzymatic active site

Klotho structure-function relationships in breast cancer

Prior to mutagenesis, constructs were transferred to a Topo plasmid to preserve

the HA-tag. The selected amino acids for substitution were chosen in order to preserve

the 3-dimensional conformation of the molecule. First, a single mutation was made at

amino acid 416 by converting Glu to Gln. FL-KL and KL1 Constructs were PCR amplified

using specific primers for mutation insertion: 5-GGA ATA TTT ATT GTG CAA AAT GGC

TGG-3 (mutation insertion) and 5-CAGGAAACAGCTATGAC-3.

Afterwards, double mutation constructs were made by adding a second mutation at

residue 240 by converting Asp to Asn. For the second mutation, site directed

mutagenesis technique was employed. Constructs were PCR amplified using specific

primers for mutation insertion: 5-

CACCATTAACAACCCCTACGTGGTGGCCTGGCACG-3 and 5-

AGGGGTTGTTAATGGTGATCCAGTACTTGACCTGACCACCG-3.

Colony assays

Two days following transfection with the indicated plasmids, G418 (750 μg/mL for

all cell lines) was added to the culture media. At day 14, cells were stained using crystal

violet (Sigma) and quantitated using ImageJ software.

Western blot analysis

Cells were harvested, lysed and total protein was extracted with RIPA buffer

(50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM

EDTA, 1 mM NaF) together with a protease inhibitor cocktail (Sigma). Lysates were

resolved on 10% SDS-PAGE and immunoblotted with the indicated antibodies. Band

intensities were quantified using ImageJ software.

Immunoprecipitation

MCF-7 cells were harvested as described above, and 400 μg protein lysate were

incubated with protein A/G (Pierce, Rockford, IL, USA) beads at 4°C for 1 hour to

eliminate nonspecifically bound proteins. These beads were subsequently used as a

Klotho structure-function relationships in breast cancer

negative control. The supernatants were then incubated with 2 mg anti-HA antibody

(Sigma) for 4 h and then protein A/G was added. Following an overnight incubation, the

beads were washed six times in RIPA buffer and the immunoprecipitated materials were

separated by SDS–polyacrylamide gel electrophoresis and detected by western blotting.

Mice tumor xenograft studies

Mice maintenance and experiments were carried out under institutional

guidelines of the Sheba Medical Center. Experiments were conducted as described (30).

Six-week-old female athymic nude mice were injected with 0.75×106 MDA-MB-231 cells

subcutaneously into both flanks. After five days, mice were treated with daily intra-

peritoneal (I.P) injections of soluble hKL1: 10 μg/kg, or saline (n = 6 per group). Tumor

size was measured weekly with a digital caliper, and the volume was estimated using the

equation V = (a × b2)×0.5236, where a is the larger dimension and b the perpendicular

diameter.

Immunohistochemistry analysis

After mice were euthanized, tumors were removed and fixed in 4%

paraformaldehyde (PFA). Paraffin-embedded tumors were serially sectioned and stained

with hematoxylin & eosin (H&E), and immunohistochemically stained for Ki-67 and

vimentin. Stained sections were examined microscopically by an experienced

pathologist. For plasma analyses, immediately after mice were euthanized, blood was

drawn from the heart and plasma was analyzed at the automated blood lab, Sheba

Medical Center.

Statistical analysis

Results are presented as mean ± SD. Continuous variables were compared

using t test. All significance tests were 2-tailed and a P < 0.05 was considered as

statistically significant.

Klotho structure-function relationships in breast cancer

Results

Structural modeling of the KL1 and KL2 domains

We generated a computational structure model of KL1 and KL2 domains based

on similarity of klotho to KLrP (Figure 1A). Crystallographic studies indicated KLrP as a

classic family 1 glycoside hydrolases that display the Koshland retaining mechanism.

Two glutamic residues serve as its catalytic residues: Glu-373 is the nucleophile and

Glu-165 is the acid/base catalyst. The model indicate that both KL1 domain (residues

57-506) and KL2 domain (residues 515-953) share significant homology to glycoside

hydrolase family 1 and are predicted to have similar TIM-Barrel fold (31) (Figure 1A).

According to the model, Glu-414 in KL1 domain can serve as the nucleophilic residue

(Figure 1B). While the corresponding residue to the acid/base catalyst Glu-165 is Asn-

239, which cannot serve as an acid/base catalyst, Asp-238 may function as an

alternative acid/base catalyst and it is in the required distance from the nucleophile (2Ǻ).

Thus, KL1 may serve as an active enzyme. In KL2, on the other hand, the critical

nucleophilic residue (Glu-373) is replaced by Ser-872, which cannot serve as the

nucleophilic residue (Figure 1C). Thus, the model suggests that KL2 is not an active

glycoside hydrolase.

KL1 inhibits colony formation and suppresses in vivo growth of breast cancer

cells

In order to evaluate the differential activities of KL1 and KL2, we constructed

expression vectors of KL1 and KL2 domain, both with a signal sequence, directing them

to the membrane (Figure 2A and B) and enabling them to be secreted (Supplementary

Fig. S1). The effect of overexpression of these domains on growth of breast cancer cells

was assessed using colony formation assays. MCF-7 and MDA-MB-231 breast cancer

cells were transfected with either klotho, KL1 or KL2 expression vectors or an empty 9

Klotho structure-function relationships in breast cancer

vector. Transfected cells were cultured in media containing G418 for two weeks and

stained to determine the number of surviving colonies. Klotho and KL1 expression

significantly reduced the number and size of surviving colonies, while KL2 showed only a

modest effect (Figure 2C and D). The tumor suppressor activity of KL1 was also verified

in vivo. For this study, MDA-MB-231 cells were injected to both flanks of athymic mice

and mice were treated with daily I.P injections of either control vehicle (n = 6) or soluble

hKL1 (10 µg/kg, n= 6), for 4 weeks. Already after 12 days of treatment, a significant

inhibition of tumor growth was noted among KL1-treated mice (p< 0.001, for comparison

of tumor volume between the control and hKL1 -treated group), lasting to the end of the

experiment (Figure 3A). Tumor weight at the end of the experiment was significantly

lower (p<0.003) in hKL1-treated group (Figure 3B and C). Immunohistochemistry

analysis revealed a significant reduction in the number of cells expressing the

proliferation marker Ki67 and the mesenchymal marker vimentin, suggesting effect of

KL1 on proliferation, as well as differentiation (Figure 3D). Importantly, KL1 treatment

was safe and did not affect the weight of mice (Figure 3A) and was associated with

higher albumin levels and reduced liver function tests and lactate dehydrogenase (LDH)

(Table 1).

Differential modulation of IGF-1 signaling by KL1 and KL2

We have previously shown that klotho efficiently suppresses activation of the

IGF-1 pathway and interacts with the IGF-1R in breast cancer cells (9). In order to

analyze the differential effects of KL1 and KL2 on IGF-1 signaling, MCF-7 cells were

transfected with either klotho, KL1 or KL2 expression vectors or an empty vector, starved

for 48 hours, treated with IGF-1 (100 ng/ml, 15 min) and analyzed using Western blotting

for the level of expression and phosphorylation of IGF-1R, AKT and ERK1/2. KL1 as well

as full length klotho reduced IGF-1-induced phosphorylation of these proteins, while KL2

Klotho structure-function relationships in breast cancer

had only minor effect (Figure 4A). Co-IP assays using MCF-7 cells expressing either

klotho, KL1 or KL2, indicated that only klotho and KL1 can interact with the endogenous

IGF-1R (Figure 4B).

Only full-length, membrane bound klotho can transduce FGF23 signaling

Membrane-bound klotho is an essential co-factor for activation of the FGFRs by

FGF23 (8). To our knowledge, the ability of KL1 and KL2 to modulate FGF23 signaling

has not been determined yet. In order to test this, MCF-7 and HEK293 cells were

transfected with either klotho, KL1 or KL2 expression vectors or an empty vector, starved

for 48 hours, treated with FGF23 (100 ng/ml, 15 min), and analyzed using Western

blotting for the level of expression and phosphorylation of ERK1/2. Neither KL1 nor KL2

were able to mediate FGF23-induced phosphorylation of ERK1/2 (Figure 4C). Removing

the IC/TM domain (FL-KL-ΔIC/TM) which anchors klotho to the cell membrane also

impaired its ability to transduce FGF23 signal (Figure 4D).

In order to test if membrane-bound KL1 can mediate FGF23 signaling we

generated a KL1 domain fused with the IC/TM region of klotho (KL1-IC/TM, Figure 2A).

HEK 293 cells were transfected with these constructs, serum starved for 48 hours,

stimulated with either a vehicle or 100ng/ml FGF23 for 15 min and analyzed using

Western blotting for the phosphorylation of ERK1/2. KL1-IC/TM failed to transduce the

FGF23 signal (Figure 4D).

Glu-416 and Asp-240 are not required for klotho tumor suppressor activity

Mutations of klotho putative active sites abolished its effect on TRPV5 and on the

renal phosphate transporter NaPi-2a (11, 32, 33) but did not affect its ability to inhibit

Wnt signaling (34). To our knowledge, the role of these residues in mediating klotho

tumor suppressor activities has not been studied yet. In order to decipher the role of

Klotho structure-function relationships in breast cancer

these amino acids in mediating the tumor suppressor activity of klotho, a series of mouse

klotho and KL1 constructs were designed, in which amino-acids have been mutated.

Glu-416 (putative nucleophile, corresponds to human Glu-414) was converted to

glutamine and Asp-240 (putative acid/base catalyst, corresponds to human Glu-238)

was converted to asparagine (Figure 1B). These mutations are not expected to alter the

tertiary structure of the protein. The ability of the various mutated proteins to inhibit

colony formation of MCF-7 and MDA-MB-231 cells was determined as described above.

In both breast cancer cell lines the mutated klotho constructs, FL-KL or KL1, harboring

single (416) or double (240-416) mutations, retained the anti-proliferative effect exhibited

by klotho. Surprisingly, the mutated klotho and KL1 proteins displayed even a more

potent anti-proliferative activity compared to their wild-type counterparts. For example in

MCF-7 cells, FL-KL, FL-KL-416 and FL-KL-240-416 reduced number of colonies to 61%,

37% and 45% of control, respectively (Figure 5A). In MDA-MB 231 cells FL-KL, FL-KL-

416 and FL-KL-240-416 reduced number of colonies to 32%, 18% and 16% of control,

respectively (Figure 5 B).

Dissecting klotho tumor suppressor activities from its physiologic activities may

enable the development of safe klotho-based therapies. We, therefore, aimed to study

whether klotho mutant, which potently slow proliferation of cancer cells, can modulate

FGF23 signaling. As only FL-KL can function as a co-factor for FGF23 (Figure 4C and

D), we analyzed only FL-KL mutants. To this aim, HEK293 cells were transfected with

either FL-KL, FL-KL-416 and FL-KL-240-416 expression vectors or an empty vector,

starved for 48 hours, treated with FGF23 (100 ng/ml, 15 min), and analyzed using

Western blotting for the level of expression and phosphorylation of ERK1/2. While the

WT-FL KL-416 was able to mediate FGF23-induced phosphorylation of ERK1/2 (Figure

5C), transfection with FL-KL-240-416 only partially increased ERK phosphorylation

(Figure 5C).

Klotho structure-function relationships in breast cancer

Discussion

This structure-function analysis of klotho in breast cancer indicates KL1 as

sufficient to mediate growth inhibition, while KL2 does not have a significant tumor

suppressor activity. Furthermore, our data suggest that the activity of KL1, as well as of

the full-length protein, is independent of the amino acids of its putative catalytic site.

Distinct activities of klotho, KL1 and KL2 have been demonstrated in several

systems. The FGF23 co-receptor function is mediated only by full length, membrane-

anchored klotho (5, 8) (Figure 3C and D). On the other hand, either KL1 or KL2 were

sufficient to activate TRPV5 (11). In contrast, several klotho activities were shown to be

KL1-dependent. Thus, it was demonstrated that KL1 is essential and sufficient to

mediate the anti-inflammatory activity of klotho in senescent cells while KL2 did not show

any such activity (35). Similarly, KL1 is mandatory and sufficient to inhibit Wnt signaling

by klotho, while KL2 is devoid of this activity (34). Our data suggest that klotho tumor

suppressor activities are KL1-dependent and that KL2 by itself cannot significantly inhibit

proliferation of breast cancer cells.

Administration of KL1 slowed tumor formation by breast cancer cells in nude

mice. Tumors from KL1-treated mice were smaller, showed reduced proliferation and

reduced expression of the EMT marker vimentin. Furthermore, their blood albumin levels

were higher and LDH levels lower compared to control-treated mice. Treatment did not

adversely affect either weight or kidney functions. This favorable toxicity profile can be

attributed to the inability of KL1 to serve as a co-receptor for FGF23. These data support

our previous observation using pancreatic cancer cells (10) and suggest that treatment

with KL1 is safe and may serve as an effective novel strategy for the treatment of breast

cancer.

Klotho structure-function relationships in breast cancer

While ample data indicate klotho as a potent tumor suppressor in a wide array of

malignancies (9, 10, 15, 16, 36), its precise mechanism of action is still a matter of

debate. Mechanisms implicated in its tumor suppressor activities include inhibition of

insulin and IGF-1 signaling, modulation of FGF signaling and inhibition of Wnt signaling

(4, 5, 8, 9, 12, 15, 37). We noted a correlation between the ability to inhibit the IGF-1

pathway, interaction with the IGF-1R and inhibition of proliferation. Thus, klotho and KL1

slowed proliferation, interacted with the IGF-1R and inhibited the IGF-1 pathway.

Conversely, KL2, which did not slow proliferation, did not inhibit the pathway or

interacted with the receptor. These data provide an indirect support to the hypothesis

that inhibition of the IGF-1 pathway is a major mediator of klotho activities in breast

cancer. Interestingly, the same klotho region required for tumor suppression is the one

required for the inhibition of the IGF-1 pathway and of Wnt signaling (10, 34). Thus, it is

possible that a common mechanism that involves inhibition of the Wnt and IGF-1

signaling pathways is responsible to klotho tumor suppressor activities.

While klotho enables activation of the FGFR pathway by FGF23, it inhibits

activation of the IGF-1 pathway. Thus, treatment with klotho can lead to either activation

or inhibition of the ERK pathway, the downstream effector of both pathways. While this

may seem to be counterintuitive, it is important to note that the activity of the ERK

pathway is tissue and cell type-dependent (38). The ERK pathway regulates a wide

array of physiologic and pathologic processes, including proliferation, development and

metabolism, and while in the kidneys, as a downstream effector of FGF23 signaling it

participates in regulating phosphorus homeostasis (39), in cancer cells it enhances

proliferation as a downstream effector of multiple growth factors, including IGF-1 (40).

Furthermore, the effect of klotho on various tyrosine kinase receptors is also complex.

While klotho enhances activation of FGFRs by FGF23 (5, 8) either klotho or KL1 inhibit

activation of the FGFRs by bFGF (10). As klotho regulates glycosylation pattern of

Klotho structure-function relationships in breast cancer

various membrane proteins (33, 41), it is possible that some of its activities are mediated

by regulating glycosylation pattern of the IGF-1R and FGFRs.

MCF-7 cells express somewhat higher klotho levels compared to MDA-MB-231

cells (9). Yet, MCF-7 cells are more sensitive to the tumorigenic effects of IGF-1 (42-45).

These data suggest a resistance to endogenous klotho in MCF-7 cells. The mechanisms

mediating this resistance to endogenous klotho remain to be elucidated. The IGF-1

signaling may play a less prominent role in MDA-MB-231 cells (44), and other signaling

pathways, including Wnt signaling, are important mediators of tumorigenesis in these

cells. As klotho is a potent inhibitor of Wnt signaling in other malignancies, it is possible

that some of its effects in breast cancer are mediating by affecting Wnt signaling (37,

46).

Several studies demonstrated a critical role for residues within klotho's putative

active site in mediating some of the activities of klotho. Thus, mutations of these

residues abolished its effect on TRPV5 and on the renal phosphate transporter NaPi-2a

(11, 32, 33) but did not affect its ability to inhibit Wnt signaling (34). Our studies indicate

that these amino acids are not required for klotho tumor suppressor activity. These data

strengthen a possible relationship between klotho tumor suppressor activity and Wnt

inhibition and suggest that enzymatic activity of klotho may not be required for its activity

against cancer cells. On the other hand, the klotho double mutant showed reduced

ability to mediate FGF23 signaling. This observation indicates that klotho's diverse

activities are mediated by different regions and may lead for the development of klotho-

based molecules showing differential activities. The design of such molecules may

enable the development of rational novel active and safe cancer therapies.

Taken together, our data indicate differential activity of KL1 and KL2 domains of

klotho in breast cancer cells and suggest that klotho tumor suppressor activities are not

mediated by enzymatic activity. Importantly, our studies indicate that it is possible to

Klotho structure-function relationships in breast cancer

dissociate klotho tumor suppressor activities from other activities of it. These

observations may pave the way for a rational design of klotho-based molecules for the

treatment of breast cancer.

Authors' Contributions

Conception and designing experiments: H. Ligumsky, K. Merenbakh-Lamin, T.

Rubinek and I. Wolf

Acquisition of data: H. Ligumsky and K. Merenbakh-Lamin.

Cloning of expression constructs: H. Ligumsky, K. Merenbakh-Lamin and S.

Shamoon.

Generation of computerized protein models: A. Yeheskel, R. Sertchook

Analysis and interpretation of data: H. Ligumsky, T. Rubinek and I. Wolf

Writing: H. Ligumsky, A. Yeheskel, T. Rubinek and I. Wolf. All authors commented on

the manuscript

Grant Support

This study was supported in part by the Israeli Science Foundation (grant number

1112/09), the Israel Cancer Association Research Grant by Peter & Nancy Brown in

memory of Eric & Melvin Brown, and the Sackler Faculty of Medicine, Tel Aviv

University, Tel-Aviv, Israel.

Klotho structure-function relationships in breast cancer

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Klotho structure-function relationships in breast cancer

Table 1: Comparison of blood chemistry parameters between the control and mice treated with soluble hKL1 (10 µg/kg) Parameter Control Soluble hKL1 p. value (N=6) (N=6) (Con. Vs. Soluble hKL1) Urea (mg/dL) 56.6 ± 5.03 57.33 ± 7.06 0.85 Creatinine (mg/dL) 0.24 ± 0.02 0.25 ± 0.02 0.46 Glucose (mg/dL) 205.2 ± 26.57 204.5 ± 24.92 0.96 Phosphorous (mg/dL) 7.8 ± 0.39 7.4 ± 0.89 0.48 Sodium (mg/dL) 142.6 ± 5.73 148 ± 2 0.057 Calcium mg/dL) 9.04 ± 0.67 9.42 ± 0.58 0.15 Chloride (mg/dL) 107.8 ± 3.83 110.67 ± 2.16 0.34 Potassium (mg/dL) 7.14 ± 1.07 6.53 ± 1.57 0.37 Uric acid (mg/dL) 1.92 ± 0.28 1.65 ± 0.43 0.25 SGOT (AST) IU/L 574.80 ± 323.23 232.67 ± 122.01 0.04* SGPT (ALT) IU/L 273.75 ± 199.85 75.83 ± 55.36 0.04* LDH IU/L 2534.33 ± 1348.54 698.75 ± 251.42 0.04* Alkaline Phosphatase 49.8 ± 14.34 81.17 ± 16.94 0.01* (IU/L) Protein (g/dL) 5.02 ± 0.5 5.53 ± 0.37 0.08 Albumin (g/dL) 2.66 ± 0.21 3.02 ± 0.29 0.05* Globulin (g/dL) 2.36 ± 0.32 2.5 ± 0.14 0.35

Table legend: Blood chemistry parameters from vehicle and soluble hKL1 treated nude mice harboring MDA-MB-231 human breast cancer cells tumors. Plasma was analyzed by automated blood lab (* p<0.05, soluble hKL1 treated mice vs control vehicle).

Figure legends

Figure 1: Structure–Function Relationships of klotho internal repeats. (A) Predicted

structure of klotho internal repeats with putative active site residues (in yellow),

responsible for the catalytic mechanism of a family 1 glycosidase. (B) Superimposition of

KLrP and KL1 active sites. KLrP active site side chains are presented in pink and KL1

domain in cyan. KLrP was crystallized with part of its product, glucose. (C)

Superimposition of KLrP and KL2 active sites. KLrP active site side chains are presented

in pink and KL2 domain in green. KLrP was crystallized with part of its product, glucose.

Klotho structure-function relationships in breast cancer

Figure 2: Klotho internal repeats exhibit differential effect on colony formation in

breast cancer cells. (A) Schematic illustration of klotho constructs used in the study (B)

MCF-7 and MDA-MB-231 cells were transfected with FL-KL and indicated truncated KL1

and KL2 HA-tagged constructs. Total cell lysates were analyzed by immunoblotting with

anti HA antibody. (C) MCF-7 and MDA-MB-231 cells were transfected with either a full-

length klotho expression vector (FL-KL), KL1, KL2 or an empty vector (pcDNA3) (all

constructs depicted in (A)). Transfected cells were cultured in media containing G418 for

two weeks. Colonies were fixed and stained with crystal violet and photographed. All

experiments were conducted at least 3 times and representative wells for each condition

are shown. (D) Quantitation of the colonies was performed using ImageJ (* p<0.05, FL-

KL and KL1 vs control).

Figure 3: Soluble human KL1 (hKL1) inhibits growth of breast tumors in nude

mice. (A) MDA-MB-231 cells were injected into both flanks of six-week-old female

athymic nude mice. The mice (N=6 per group) were treated with daily intra-peritoneal

injections of soluble hKL1 (10 μg/kg) or vehicle control (saline), for 3 weeks. Tumors

volumes were measured weekly (**P<0.001, for tumor volume between soluble hKL1

treated and control group). (B) Representative photographs of mice with tumors in

soluble hKL1 treated group and control group before they were sacrificed. (C) Tumors

weight was measured on day of sacrifice (**P < 0.01 for comparison of tumor weight

between the control group and the mice treated with soluble hKL1). (D) H&E, Ki67,

vimentin and cytokeratin AE1/AE3 immunostaining (x400 magnification). (E). Mice

weight of soluble hKL1 treated group and control group at the beginning and the end of

the I.P injections.

Klotho structure-function relationships in breast cancer

Figure 4: Only KL1 domain suppresses IGF-1 signaling and interacts with the IGF-

1R in breast cancer cells. (A) MCF-7 cells were transfected as described above,

starved for 48 hours and then stimulated with IGF-1 for 15 min. Cell lysates were

immunoblotted using anti-phosphorylated (p) IGF-1R, p- and total (t)-AKT and p- and t-

ERK1/2 antibodies. Phosphorylated to-total ratio is calculated relative to untreated

pcDNA3. Band quantification was performed using ImageJ. (B) MCF-7 cells over-

expressing full length klotho (FL-KL) KL1 and KL2 or a control vector were lysed and 400

μg protein was immunoprecipitated for total IGF-1R with anti-IGF-1R antibody. After

addition of protein A/G, immunocomplexes were washed, resolved on SDS–

polyacrylamide gel electrophoresis gels and immunoblotted with anti-HA antibody.

Preliminary unprocessed cell lysate (input) is also shown. (*IgG heavy chain). (C) MCF-7

and HEK-293 were transfected as described above, starved for 48 hours and then

stimulated with FGF23 (100 ng/ml). Cell lysates were immunoblotted using phospho (p)

and total (t) ERK1/2. (D) HEK-293 cells were transfected with either FL-KL, FL-KL

without IC/TM, pEF (vector control), KL1, KL1 with IC/TM or pcDNA3 (vector control).

Following 48 hours serum starvation, cells were stimulated with vehicle or 100ng/ml

FGF23 for 15 min and snap-frozen in liquid nitrogen. Cell lysates were prepared for

Western blot using antibodies against phosphorylated ERK1/2 (pERK1/2) and total

ERK1/2.

Figure 5: Mutating klotho putative enzymatic active site does not affect klotho

anti-cancerous activity while compromising FGF23 signaling. (A, B) MCF-7 and

MDA-MB-231 cells were transfected as described above. Transfected cells were

cultured in media containing G418 for two weeks. Colonies were fixed and stained with

crystal violet and photographed. All experiments were conducted at least 3 times and

representative wells for each condition are shown. Quantitation of the colonies was 22

Klotho structure-function relationships in breast cancer

performed using ImageJ (* p<0.05, FL-KL, FL-KL-416, FL-KL-240-416, KL1, KL1-416

and KL1-240-416 vs matched control; # p<0.05, FL-KL-416, FL-KL-240-416 vs WT FL-

KL and KL1, KL1- 416 and KL1-240-416 vs WT KL1). (C) HEK 293 cells were

transfected with either FL-KL, FL-KL-416, FL-KL-240-416, KL1, KL1-416 and KL1-240-

416 or pcDNA3 (vector control). Following 48 hours serum starvation, cells were

stimulated with vehicle or 100ng/ml FGF23 for 15 min and snap-frozen in liquid nitrogen.

Cell lysates were prepared for Western blot using antibodies against phosphorylated

ERK1/2 (pERK1/2), and total ERK1/2.

Figure 1 A

Figure 1

B C KL1 KL2

Figure 2 A B

C D

* * * *

Figure 3 A B C

D E

Figure 4 A B

C D

Figure 5

A B FL-KL KL1

Figure 5

pcDNA3 FL-KL FL-KL-416 FL-KL-240-416 FGF23 100ng/ml - + - + - + - + pERK1/2

tERK1/2

Tumor Suppressor Activity of Klotho in Breast Cancer is Revealed by Structure-function Analysis

Hagai Ligumsky, Tami Rubinek, Keren Merenbakh-Lamin, et al.

Mol Cancer Res Published OnlineFirst June 25, 2015.

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