2024.01.21.576539v1.full-html.html

bioRxiv preprint

STRACT

Cancer initiation and progression result from both genetic alterations and epigenetic

reprograming caused by environmental or endogenous factors which can lead to

aberrant cell signalling. Most colorectal cancers (CRC) are linked to the abnormal

activation of the Wnt/

-catenin pathway, whose key feature is the accumulation of

acetylated

-catenin protein within the nucleus of colon epithelial cells. Nuclear

catenin acts as a transcriptional co-activator that alters the expression of many target

genes involved in cell proliferation and invasion. The most active vitamin D metabolite

1,25-dihydroxyvitamin D

(1,25(OH)

calcitriol) can antagonize the over-activated

Wnt/

-catenin pathway via binding to its high affinity receptor VDR. Here, we show

that the activation of the SIRT1 deacetylase by 1,25(OH)

-bound VDR promotes

deacetylation and nuclear exclusion of

-catenin and, consequently, the

downregulation of its pro-tumorigenic target genes and the inhibition of human colon

carcinoma cell proliferation. Notably, orthogonal SIRT1 activation systematically

drives nuclear exclusion of

-catenin, highlighting the key role of SIRT1 in CRC.

Since nuclear localization of

-catenin is a critical driver of CRC initiation and

progression that requires its acetylation, our results provide a mechanistic basis for the

epidemiological evidence linking vitamin D deficiency and increased CRC risk and

mortality.

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INTRODUCTION

Epidemiological studies suggest that vitamin D (cholecalciferol, VD) deficiency

may be a risk factor for developing and dying of cancer, particularly for

colon/colorectal cancer (CRC) [1–4]. However, data from supplementation studies in

the human population are controversial, and confirmation of a clinically relevant anti-

CRC effect of VD in well-designed prospective randomized trials is pending [5–9].

Post-hoc analyses indicate that this discrepancy may rely on the lack of patient

stratification in the clinical trials, suggesting that VD intervention is effective in the

deficient/insufficient VD subjects but not in VD sufficient individuals, and is also

dependent on conditions such as patient body mass, ethnicity, mutational status, or

genotype of VD-related genes [6,10,11]. Importantly, many mechanistic experimental

studies show a wide range of antitumoral effects of VD in CRC and other neoplasia

that strongly support a protective VD action [12–14].

VD is incorporated in humans through diet or is synthesized in the skin by solar

ultraviolet radiation-dependent transformation of 7-dehydrocholesterol. The active VD

metabolite is 1

,25-dihydroxyvitamin D

(1,25(OH)

, calcitriol, which results from

two consecutive hydroxylation of VD, the first in the liver and the second in the kidney

or in many epithelial and immune cell types in the organism [1,15].

1,25(OH)

binds

to a member of the nuclear receptor superfamily of transcription factors, the vitamin

D receptor (VDR). Upon ligand binding, VDR regulates the pleiotropic actions of VD

including its many anticancer effects on cell survival, proliferation and differentiation

[1,13,15–17].

The anti-CRC properties of liganded VDR largely rely on its capacity to interfere

with a well-recognized CRC driver, the canonical Wnt/

-catenin signalling pathway

that regulates many protumoral genes and which is abnormally over-activated in most

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bioRxiv preprint

CRC [18]. Consistently, germline

Vdr

deletion in

Apc

Min/+

mice increases Wnt/

catenin signalling activation and intestinal tumour load [19,20]. From the many

possible steps to block Wnt/

-catenin signalling, the most precise is acting directly on

-catenin, its downstream transcriptional effector. Acetylation of

-catenin promotes

its nuclear localization and transcriptional activity in cancer cells [21–23].

-catenin

acetylation is increased in CRC cells by the combination of Wnt signalling and

enhanced glucose uptake. The coincidence of Wnt and high glucose increases the

levels and activity of the acetyl transferase EP300 and inhibits SIRT1-driven

deacetylation of

-catenin without changing SIRT1 levels [21] by mechanisms that

remain unknown.

Since 1,25(OH)

induces SIRT1 activity in CRC cells [24], we investigated

whether 1,25(OH)

impedes the Wnt/

-catenin pathway via regulation of SIRT1. In

particular, we examined the intriguing possibility that 1,25(OH)

impacts on CRC

by blocking the glucose-driven

-catenin acetylation that enhances Wnt/

-catenin

signalling.

RESULTS

1,25(OH)

reverses the acetylation of SIRT1 promoted by Wnt and induces

catenin nuclear export in CRC cells.

1,25(OH)

-bound VDR can induce SIRT1 auto-deacetylation and activation [24].

Since SIRT1 targets nuclear exclusion of

-catenin in cancer cells [21] and

1,25(OH)

interferes with Wnt/

-catenin signalling, we first asked whether SIRT1

mediates this effect of 1,25(OH)

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SIRT1 acetylation is a hallmark mark of its inactivity [24]. Given the

100

importance of SIRT1 acetylation for its activity, we studied whether Wnt signals could

101

affect the acetylation status of SIRT1 in nuclear extracts of CRC cells. Exposure of

102

cells to Wnt3A, or LiCl to mimic Wnt signaling, strongly induced SIRT1 acetylation

103

that was reversed by treatment with 1,25(OH)

(Figure 1A). Additionally,

104

1,25(OH)

increased SIRT1 levels in CRC cells independently of the presence of

105

Wnt signals (Figure 1

). Thus, 1,25(OH)

ensures SIRT1 induction by increasing

106

both its levels and its deacetylation. Interestingly, co-immunoprecipitation assays

107

demonstrated that 1,25(OH)

favored SIRT1/

-catenin interaction (Figure 1C).

108

Consistently, 1,25(OH)

promoted

-catenin deacetylation in CRC cells (Figure

109

1D), which paralleled

-catenin nuclear exclusion examined by western blotting

110

(Supplementary Figure S1A) and immunofluorescence analyses (Figure 1E and

111

Supplementary Figure S1

). Accordingly, 1,25(OH)

reduced the expression of

112

catenin target genes associated with tumor progression such as

MYC

and

CCDN1

113

(Figure 1F, 1G and Supplementary Figure S1C). Notably, 1,25(OH)

could interfere

114

with Wnt signalling independently of whether it was added before or after Wnt

115

activation (Supplementary Figure S1D), suggesting a potential for both prevention and

116

treatment.

117

118

SIRT1 activity inversely correlates with the level of nuclear

-catenin during human

119

CRC progression.

120

The striking reversal of the Wnt effect on SIRT1 and

-catenin exerted by 1,25(OH)

121

in CRC cells led us to investigate whether SIRT1 levels were related to

-catenin

122

localization in human colorectal tumors, in which nuclear accumulation of

-catenin

123

is a marker of poor prognosis. Human tissue microarrays containing samples (n = 109)

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from primary tumors of stage II and stage IV patients and from liver metastasis of the

125

latter were examined to understand the relationship between SIRT1 level and activity

126

and the subcellular localization of

-catenin. SIRT1 activity was inferred by evaluating

127

the acetylation of its specific well characterized substrate, lysine 9 of histone 3

128

(AceH3K9).

129

Although the level of SIRT1 did not change (Figure 2A), AceH3K9 levels, a mark for

130

SIRT1 inactivity increased significantly from primary tumors of stage II patients to

131

metastasis (

= 0.027) (Figure 2B). Accordingly, nuclear

-catenin level increased

132

(Figure 2C) while that of cytoplasmic

-catenin was reduced (Figure 2D) through

133

tumor progression as expected.

134

Interestingly, a significant positive association (

= 0.003) (Figure 2E) and

135

correlation (R = 0.334,

< 0.001) (Figure 2F) between SIRT1 protein levels and

136

cytoplasmic

-catenin levels were found. In addition, significant positive correlations

137

between these parameters were also observed considering separately primary tumors

138

from stage II patients (R = 0.272,

< 0.027), primary tumors from stage IV patients

139

(R = 0.622,

= 0.008) and liver metastases (R = 0.480,

= 0.013) (Figure 2F). Thus,

140

human CRC samples with a low level of SIRT1 tend to have a high level of nuclear

141

catenin and

vice versa

. Representative photographs show the coincidence of low

142

SIRT1 level with high nuclear

-catenin in consecutive sections. Samples with high

143

SIRT1 level tend to have high cytoplasmic

-catenin (Figure 2G), although there were

144

samples with high level of SIRT1 and nuclear

-catenin, which could be explained if

145

SIRT1 activity was diminished, as it seems to happen by analysing their high AceH3K9

146

level (Figure 2H). Collectively, these results suggest that decreased SIRT1 activity

147

(independently of its levels) drives a failure to export

-catenin from the nucleus,

148

which may worsen CRC prognosis.

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SIRT1 is required for nuclear exclusion of

catenin by 1,25(OH)

150

To understand whether the nuclear export or accumulation of

-catenin was caused by

151

SIRT1 induction or inhibition, we modulated SIRT1 activity. Treatment with the

152

general sirtuin inhibitor nicotinamide (NAA) prevented the nuclear depletion of

153

catenin induced by 1,25(OH)

in CRC cells as analysed by immunofluorescence

154

(Figure 3A) and western blotting (Figure 3

and Supplementary Figure S2A). Similar

155

results were obtained with the more specific SIRT1 inhibitor EX527 (Figure 3C).

156

SIRT1 was also depleted using several siRNAs (Supplementary Figure S2

) and its

157

depletion also blocked the 1,25(OH)

-mediated nuclear depletion of

-catenin

158

(Figure 3D and Supplementary Figure S2C). In line with these results, SIRT1

159

inhibition with EX527 abolished the antiproliferative effect of 1,25(OH)

on CRC

160

cells (Figure 3E).

161

Conversely, the specific activation of SIRT1 by the small molecule SRT1720 drove

162

nuclear exclusion of

-catenin, as shown by immunofluorescence (Figure 3F) or by

163

western blotting (Figure 3G). Similar antiproliferative effects were obtained by

164

activation of SIRT1 with either 1,25(OH)

or SRT1720 (Figure 3H). These results

165

show that the 1,25(OH)

-driven depletion of nuclear

-catenin that interferes Wnt

166

signalling is critically mediated by SIRT1 activity.

167

A constitutively active SIRT1 mutant mimics the reduction of nuclear

-catenin by

168

1,25(OH)

169

Given the role of SIRT1 on the subcellular localization of

-catenin, we exogenously

170

expressed SIRT1 to ask whether it mediates 1,25(OH)

action controlling

-catenin

171

acetylation and localization. To this end, Myc-tagged SIRT1 wild-type (WT), or the

172

inactive H363Y [25] or the constitutively active K610R [24] SIRT1 mutants were

173

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expressed to similar levels in CRC cells. Nuclear extracts from cells transfected with

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SIRT1 WT or mutants were immunoprecipitated using anti-acetyl-lysine antibodies

175

and subjected to western blotting to evaluate the extent of

-catenin acetylation. In the

176

nuclei of CRC cells, acetylated

-catenin decreased upon expression of WT SIRT1,

177

increased after expression of the inactive H363Y mutant and was lost upon expression

178

of the constitutively active K610R mutant (Figure 4A). Consistently, the nuclear levels

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-catenin reflected its acetylation status and was reduced by half after expression of

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WT SIRT1, whereas the inactive H363Y mutant had no effect, and nuclear

-catenin

181

was almost completely depleted (4-5-fold reduction) following expression of the

182

constitutively active K610R SIRT1 (Figure 4

183

We also examined

-catenin subcellular localization in response to

184

1,25(OH)

upon expression of these mutants. 1,25(OH)

treatment in control cells

185

reduced the level of nuclear

-catenin up to 50%, a small reduction was measured in

186

cells expressing WT SIRT1, whereas in cells expressing the constitutively active

187

K610R SIRT1 nuclear

-catenin maximally diminished by 75% regardless of

188

1,25(OH)

treatment (Figure 4C). Conversely, cells expressing the inactive H363Y

189

SIRT1 mutant exhibited higher basal nuclear levels of

-catenin and were also

190

unresponsive to 1,25(OH)

(Figure 4C). Of note, immunofluorescence analyses

191

confirmed that K610R SIRT1 was constitutively active and depleted

-catenin from

192

the nucleus of HCT 116 cells while, in contrast, the inactive H363Y SIRT1 mutant led

193

to nuclear accumulation of

-catenin (Figure 4D). Collectively, our data indicated that

194

1,25(OH)

induces SIRT1 activity, which controls

-catenin acetylation, subcellular

195

localization, and transcriptional activity.

196

SIRT1 activity offers a surrogate target to inhibit the Wnt/

-catenin pathway in

197

cases of 1,25(OH)

unresponsiveness.

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VDR expression is the main determinant of cell responsiveness to 1,25(OH)

199

and is often downregulated in advanced CRC, which together with frequent VD

200

deficiency implies that these patients would probably not benefit from the anticancer

201

effects of 1,25(OH)

[20,26–28]. We sought to investigate the potential of targeting

202

SIRT1 under these conditions by using HCT 116 cells in which VDR expression was

203

downregulated by means of shRNA[19]. Western blotting and immunofluorescence

204

analyses showed that the nuclear content of

-catenin was slightly higher in ShVDR

205

cells, which did not respond to 1,25(OH)

by excluding

-catenin from their nuclei

206

as expected. In contrast, ShControl cells responded to 1,25(OH)

with strong nuclear

207

-catenin depletion (Figure 5A-B).

208

Next, we examined the potential to overcome the lack of response to

209

1,25(OH)

in ShVDR HCT116 cells by expressing the SIRT1 mutants (Figure 5C).

210

Interestingly, expression of the constitutively active K610R SIRT1 mutant reduced

211

nuclear

-catenin levels in both ShControl and ShVDR cells to an extent similar to

212

that achieved by 1,25(OH)

treatment in ShControl cells. As expected, the inactive

213

H363Y SIRT1 did not modify nuclear

-catenin levels (Figure 5C). These results

214

suggested that targeting SIRT1 activity might alleviate the effects of VD deficiency or

215

unresponsiveness. Indeed, the specific SIRT1 activator SRT1720 reduced nuclear

216

catenin levels in both ShControl and ShVDR cells, as shown in nuclear extracts by

217

western blotting (Figure 5D) or in whole cells by immunofluorescence analysis (Figure

218

5E).

Accordingly, SRT1720 reduced the levels of Wnt target proteins such as MYC and Cyclin

219

D1 in ShControl cells at a comparable magnitude to 1,25(OH)

Notably, SRT1720 also

220

reduced MYC and Cyclin D1 protein levels in the vitamin D unresponsive ShVDR cells

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(Figure 5F)

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These results indicate that Wnt/

-catenin signalling can be reduced by targeting SIRT1

223

in cells unresponsive to 1,25(OH)

. Altogether, this work demonstrates that

224

1,25(OH)

(i) induces reversal of SIRT1 acetylation imposed by Wnt, (ii) favors

225

SIRT1/

-catenin interaction, (iii) increases

-catenin deacetylation, (iv) promotes

226

nuclear exclusion of

-catenin and (v) decreases expression of key Wnt-target genes

227

(Figure 5G).

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229

DISCUSSION

230

Here we reveal that SIRT1 activity is a critical mediator of the anticancer properties of

231

1,25(OH)

related to its interference of Wnt/

-catenin signalling in CRC. The

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critical importance of Wnt signalling in CRC is emphasized by the fact that over 94%

233

of primary and up to 96% of metastatic colorectal tumors contain mutations that

234

aberrantly activate Wnt/

-catenin signaling [29,30] Importantly, Wnt/

-catenin

235

induces a gene signature (largely coincident with that of intestinal stem cells) that

236

identifies CRC stem cells and predicts disease relapse [31]

237

Previous work of our group and others has unveiled 1,25(OH)

targets that

238

interfere Wnt/

-catenin signalling, such as VDR-dependent increased E-cadherin

239

expression and improved E-cadherin-

-catenin interactions at the membrane [32],

240

competition between VDR and Tcf/Lef transcription factors to bind

-catenin

241

[12,13,33], and the induction of the Wnt inhibitor DKK-1 [34]. This work

242

demonstrates that inhibition or depletion of SIRT1 leaves 1,25(OH)

unable to

243

interfere Wnt target gene expression or proliferation in CRC cells, thus providing a

244

mechanism that mediates previously described effects.

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Surely, acetylation governs protein-protein and protein-DNA interactions due to

246

increased size of the lysine side chain and neutralization of its positive charge,

247

potentially disrupting salt bridges and introducing steric bulks [35–37]. Indeed, VDR

248

deacetylation by SIRT1 increases its transcriptional activity on specific genes [38] and

249

may as well drive increased expression of alternative VDR target genes capable to

250

interfere Wnt signalling such as E-cadherin or DKK1. In fact, we observe E-cadherin

251

protein levels regulated through SIRT1 modulation. Moreover, SIRT1-driven

252

deacetylation of

-catenin may establish the mark for its preference to bind VDR

253

instead of Tcf/Lef in the nucleus, its capacity to be exported and its interactions with

254

E-cadherin at the plasma membrane. Not surprisingly, there is an increasing interest

255

on the effects of vitamin D on SIRT1 [39], and on the modulation by 1,25(OH)

256

VDR and SIRT1 interactions with Fox O and NF-

B as mediators of vitamin D

257

pleiotropism [40,41].

258

The role of SIRT1 in cancer is controversial and complicated given the multiplicity of

259

its interactions with histones, transcriptional regulators and enzymes and its control of

260

genome stability, cellular differentiation, growth, and metabolism [42,43].

261

Although[44] reported that SIRT1 inhibition reduces tumorigenesis in animals,

262

suggesting that SIRT1 acts as tumor promoter, [45] reported that SIRT1-deficient

263

embryos exhibit genomic instability and [46] showed that SIRT1 suppressed tumor

264

growth in a mouse colon cancer model driven by Wnt/

-catenin. This last model

265

suggests that the tumor suppressive action of SIRT1 must be exerted on Wnt/

-catenin.

266

Notably, SIRT1 levels and activity are not regulated in parallel and there are CRCs

267

with high SIRT1 levels and low SIRT1 activity [24]. Therefore, reports on increased

268

SIRT1 levels through tumor progression, for example [47], might demonstrate that

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SIRT1 activity also increases. This is also important to interpret the correlation

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between SIRT1 overexpression in xenografts and increased tumor size since increased

271

SIRT1 levels may not correlate with increased SIRT1 activity.

272

Importantly, by using the specific SIRT1 activator SRT1720, the SIRT1

273

inhibitors NAA and EX527, several siRNAs against SIRT1, and overexpression of

274

SIRT1 WT and constitutive active K610R and inactive H363Y mutants, we

275

convincingly show that the depletion of nuclear

-catenin and the antiproliferative

276

effects exerted by 1,25(OH)

in CRC cells rely on SIRT1 induction and subsequent

277

-catenin deacetylation. Notably, we also show that the reported inactivation of SIRT1

278

by Wnt signalling [21,22] correlates with SIRT1 acetylation that is reversed by

279

1,25(OH)

treatment, reported to activate SIRT1[24] .

280

Increased nuclear

-catenin in HeLa cells upon SIRT1 overexpression [48], led

281

to conclude that SIRT1 deacetylation of

-catenin promotes its nuclear entry. This is

282

at odds with the opposite phenotype reported in this paper and with the established

283

general assumption that

-catenin acetylation improves its stability by inhibiting its

284

ubiquitin-mediated degradation and promotes its nuclear translocation [21,49–52].

285

Nonetheless, HeLa cells under basal conditions showed abundant SIRT1 with most

286

catenin in the cytosol and although nude mice with HeLa xenografts overexpressing

287

SIRT1 developed bigger tumors, this could be achieved independently of Wnt

288

signalling given the pleiotropic effects of SIRT1 on genome stability, metabolism, and

289

general transcription. In addition, SIRT1 acts at many levels on Wnt signalling

290

including reduced expression of Dvl [53] and silencing Wnt antagonists such as

291

DACT1 or sFRPs [54]. In contrast to these studies, our work shows unequivocally that

292

cells expressing constitutively active SIRT1 K610R mutant exhibit nuclei without

293

catenin, whereas cells expressing the inactive H363Y mutant accumulate nuclear

294

catenin. In addition, we show nuclear

-catenin depletion and deacetylation under

295

SIRT1 activation or over expression.

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Importantly, several acetylation sites present in

-catenin (including K19, K49,

297

K345, K354) and targeted by different acetyl transferases (C

P, EP300, pCAF),

298

govern its affinity for different proteins and may do so in a cell type specific manner.

299

This may offer an explanation to the distinct outcomes of

-catenin deacetylation in

300

distinct cell types. Indeed, acetylation of

-catenin K354 was previously reported to

301

regulate cytoplasm-nuclear shuttling of

-catenin in CRC cells [21]; however, studies

302

that suggest SIRT1 driven nuclear localization of

-catenin in other cells by

303

deacetylation are based on exogenous expression of

-catenin K49 mutants.

304

Our results show that activation of SIRT1 by 1,25(OH)

reverses the effect

305

of Wnt, excluding

-catenin from the nuclei of CRC cells. VD deficiency is strongly

306

associated epidemiologically to CRC but also to diabetes [12,13,55]. Since the effects

307

described here reverse the potentiation of Wnt signalling exerted by high glucose in

308

diabetes [22,56–60], our results suggest that VD deficiency may be one underlying

309

cause connecting these two diseases.

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Wnt signalling regulates development and drives adult tissue stem cell

311

maintenance. Although its malfunction is involved in cancer and many other diseases,

312

it is therapeutically underexploited due to toxicity and limited efficacy [61,62].

313

Unfortunately, despite the potential of 1,25(OH)

to interfere Wnt/

-catenin

314

signalling, advanced CRCs often loss VDR expression and become unresponsive.

315

Here, we highlight that, downstream of VDR, small molecule activators for SIRT1

316

such as SRT1720 offer an alternative approach and a therapeutic hope for these cancers

317

if they could be directed specifically to cancer cells. Our results point to SIRT1 as a

318

critical effector of 1,25(OH)

and rise the hope that acting on SIRT1 may circumvent

319

1,25(OH)

unresponsiveness derived from VD deficiency or VDR loss as it is

320

common in advanced CRC.

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MATERIALS AND METHODS

323

Table 1. Key Materials & Resources-

324

Key resources

325

Reagent or Resource

Source

Identifier

Antibodies

Rabbit Polyclonal anti-Acetylated-lysine

Cell Signaling

Cat# 9441

Rabbit Polyclonal anti-

-catenin (C18)

Santa Cruz

iotechnology

Cat# SC-1496-R

Mouse monoclonal anti-

-Catenin

Cat# 610154

Rabbit Polyclonal anti-SIRT1

Santa Cruz

iotechnology

Cat# SC-15404

Rabbit Polyclonal anti-SIRT1 (D1D7)

Cell Signaling

Cat# 9475

Rabbit monoclonal anti-Histone H3K9-Ace

(C5

11)

Cell Signaling

Cat # 9649

Rabbit Polyclonal anti-Histone H3

Cell Signaling

Cat # 9715

Rabbit Polyclonal anti-Vitamin D3 receptor

(D2K6W)

Cell Signaling

Cat #12550

Rabbit Polyclonal anti-TFIID-T

P (N12)

Santa Cruz

iotechnology

Cat# SC-204

Mouse monoclonal anti-T

P (51841)

Invitrogen

Cat# MA514739

Rabbit monoclonal anti-FoxO3a (75D8)

Cell Signaling

Cat# 2497

Rabbit Polyclonal anti-ERK (C14)

Santa Cruz

iotechnology

Cat# SC-154

Rabbit monoclonal anti-LEF1 (C12A5)

Cell Signaling

Cat# 2230

Goat Polyclonal antiLamin

(C20)

Santa Cruz

iotechnology

Cat# SC-6216

Mouse monoclonal anti-Lamin A/C (4C11) Cell Signaling

Cat# 4777

Mouse Monoclonal anti-MYC (9E10)

Santa Cruz

iotechnology

Cat# SC-40

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Mouse Monoclonal anti-Tubulin

-5-1-2

Sigma-Aldrich

Cat# T5168

Mouse Monoclonal anti-GAPDH

Sigma-Aldrich

Cat# G8795

Goat Anti-Rabbit IgG (H+L) HRPO

IO-RAD

Cat #170-6515

Goat Anti-Mouse IgG (H+L) HRPO

IO-RAD

Cat #170-6516

Rabbit-Anti Goat IgG (H+L) HRPO

IO-RAD

Cat #172-1034

Donkey Anti-Rabbit- AlexaFluor488

Invitrogen

Cat# A21206

Donkey Anti-Mouse- AlexaFluor488

Invitrogen

Cat# A21202

Donkey Anti-Goat-AlexaFluor647

Invitrogen

Cat# A21447

acterial and Virus Strains

E. coli 5α

New England

iolabs NE

5α

Chemicals, Peptides, and Recombinant Proteins

1α,25

-DihydroxyVD

Sigma-Aldrich

17936

Lithium Chloride

Sigma-Aldrich

Cat# L9650

Recombinant Murine Wnt-3a

Peprotech

Cat# 315-20

Glucose

Sigma-Aldrich

G8769

Sigma-Aldrich

A7906

Nicotinamide (NAA)

Sigma-Aldrich

N3376

SRT1720

Selleckchem

S1129

TRIzol reagent

Invitrogen

Cat#15596026

Protease inhibitor Cocktail

Roche

Cat#04693132001

7-AAD

Santa Cruz

iotechnology

SC-221210

3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazol

(MTT)

Sigma-Aldrich

Cat# M5655

DMEM media

Lonza

Cat# 12-604F

ovine Fetal Serum

Sigma

Cat# F7524

JetPEI PolyPlus reagent

Genycell

iotech

Cat# 101-10N

JetPRIME PolyPlus reagent

Genycell

iotech

Cat # 114-01

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QuickChange Site-Directed Mutagenesis

Kit

Stratagene

Cat #200523

SIRT-Glo Assay kit

Promega

G6450

DYNAbeads Protein A

Invitrogen

Ref 10002D

DYNAbeads Protein G

Invitrogen

Ref 10004D

Experimental Models: Cell Lines

HCT 116 (Male) colorectal carcinoma

ATCRC

Cat# CRCL-247,

RRID:CVCL_029

HT-29 (Female). Rectosigmoid

adenocarcinoma

ATCRC

Cat# HT

Β−

38,

RRID:CVCL_032

HCT 116 ShControl

In house

(Larriba et al.

2011)

HCT 116 ShVDR

In house

(Larriba et al.

2011)

Oligonucleotides

Human SIRT1 K610R mutagenesis primers

F 5

′

-3

′

GGTTCTAGTACTGGGGAGAGGAATG

AAAGAACTT CAGTGG

R 5

′

-3

′

CRCAGCRCACTGAAGTTCTTTCATTC

RCTCTCRCCRCAGT ACTAG

Sigma

This study

Human CYCLIN D1 (CRCND1) qPCR

primers

F 5

′

-3

′

: AAGATCGTCGCRCACRCTGG

R 5

′

-3

′

GGAAGACRCTCRCTCRCTCGCAC

Sigma

This study

Human c-MYC qPCR primers

F 5

′

-3

′

CTTCTCTCRCGTCRCTCGGATTCT

R 5

′

-3

′

GAAGGTGATCRCAGACTCTGACRCT

Sigma

This study

Human 18s qPCR primers

F 5

′

-3

′

AGTCRCCTGCRCCTTTGTACACA

R 5

′

-3

′

GCRCTCACTAAACRCATCRCAATCG

Sigma

This study

CYP24A1 TaqMan® probe

Applied

iosystems

Hs00167999_m1

Sirt1 siRNA (h), 10µM

Santa Cruz

iotechnology

sc-45313

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

siRNA Control

Qiagen

Cat# 1027281

Recombinant DNA

pcDNA3.1-SIRT1-Myc-His

Gift (Prof. C. Goding) N/A

pcDNA3.1-SIRT1H363Y- Myc-His

Gift (Prof. C. Goding)

N/A

pcDNA3.1-SIRT1 K601R- Myc-His

This study

N/A

Software and Algorithms

LAS AF software

Leica

SP5

3730xl Analyzer

Applied

iosystem

I 3730XL

GraphPad Prism software

GraphPad Software

https://www.grap

hpad.com

Typhoon scanner control 3.0

Applied

iosystem

Typhoon 9210

ImageLab

io-Rad

ChemiDoc XRS+

System

CXP software

ecton-Dickinson

FACSCalibur

ImageJ software

https://imagej.nih.gov/

ij/download.html

N/A

SPSS Statistics version 26

N/A

326

Table 2.

Clinicopathological characteristics of CRC patients included in the

327

study.

328

Patients Characteristic

N (%)

Stage II

Stage IV

Gender

Male

57 (60%)

27 (48%)

Female

38 (40%)

23 (41%)

N/A

6 (11%)

Tumor Location

Cecum

13 (14%)

3 (5%)

Right

25 (26%)

10 (18%)

Transverse

7 (8%)

3 (5%)

Left

5 (5%)

2 (4%)

Sigma

24 (25%)

21 (38%)

Rectum

21 (21%)

12 (21%)

N/A

5 (9%)

Grade Primary Tumor

Well differentiated

18 (19%)

15 (27%)

Moderately differentiated

69 (73%)

27 (48%)

Poorly differentiated

8 (8%)

5 (9%)

N/A

9 (16%)

Paired Liver Metastasis

54 (96%)

N/A

2 (4%)

Grade Metastasis Tumor

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this version posted January 24, 2024.

bioRxiv preprint

Well differentiated

12 (21%)

Moderately differentiated

29 (52%)

Poorly differentiated

4 (7%)

N/A

11 (20%)

Metastasis at diagnosis

Synchronous

35 (62%)

Metachronous

21 (38%)

3 (3%)

2 (4%)

30 (32%)

5 (9%)

61 (64%)

42 (75%)

1 (1%)

4 (7%)

N/A

3 (5%)

95 (100%)

27 (48%)

16 (29%)

9 (16%)

1 (2%)

N/A

3 (5%)

329

330

METHOD DETAILS

331

Colorectal cell panels

332

Human colorectal adenocarcinoma HT-29 and HCT 116 and HCT 116 derived ShControl

333

and ShVDR cells were cultured in 5%CO2 at 37ºC with DMEM containing 25 mM glucose

334

(unless specifically indicated) supplemented with 10% foetal bovine serum (FBS) and 1%

335

penicillin-streptomycin. Cells were treated as indicated for 24h. HCT 116 Sh Control and

336

ShVDR cell lines were derived previously [19]

337

Transient transfections

338

For plasmid transfection

, c

ells

were seeded in plates at 50% confluence using JetPei

339

PolyPlus reagent, following the manufacturer’s instructions. After 24h cells were treated as

340

indicated for 24 hours.

341

For Sirt1 siRNAs, cells plated in six well plates at 50% confluence were transfected with

342

JetPRIME following the manufacturer's instructions. After 2 days cells were treated 24h with

343

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bioRxiv preprint

LiCl 40 mM and then another 24h with 1,25(OH)

before being collected to analyse by

344

western blot.

345

Preparation of cell extracts:

346

Whole cell extracts.

347

Cells were washed with iced PBS before extract preparation and scraped in RIPA buffer (10

348

mM Tris HCl [pH 7.4], 5 mM EDTA, 5 mM EGTA, 1% Tryton X100, 10 mM Na

[pH

349

7.4], 10 mM NaF, 130 mM NaCl, 0.1% SDS, 0,5% Na-deoxycholate). After 5 min on ice,

350

cells were pelleted (12000 rpm for 5 min, 4ºC) and the supernatant was directly used as whole

351

cell extract or frozen at -80 ºC.

352

Fractionated cell extracts.

353

After washing as before, cells were scraped in hypotonic buffer (20 mM Hepes, [pH 8.0], 10

354

mM KCl, 0,15 mM EDTA, 0,15 mM EGTA, 0,05% NP40 and protease inhibitors) and

355

incubated on ice for 10 min before adding 1:2 vol of sucrose buffer (50 mM Hepes [pH=8.0],

356

0.25 mM EDTA, 10 mM KCl, 70% sucrose). Lysates were fractionated (5000 rpm for 5 min

357

at 4ºC) to obtain the cytoplasmic fraction in the supernatant. Nuclear pellets were washed

358

twice with washing buffer (20 mM Hepes [pH 8.0], 50 mM NaCl, MgCl

1.5 mM, 0,25 mM

359

EDTA, 0,15 mM EGTA, 25% glycerol and protease inhibitors), pelleted at 5000 rpm, 5 min

360

at 4ºC and resuspended in nuclear extraction buffer (20 mM Hepes[pH 8.0], 450 mM NaCl,

361

MgCl

1.5 mM, 0,25 mM EDTA, 0,15 mM EGTA, 0,05% NP40, 25% glycerol and protease

362

inhibitors) before centrifugation at 12,000 rpm for 5 min at 4ºC to pellet and discard cell

363

debris. The supernatants were used as nuclear fractions.

364

Immunoprecipitation

365

For immunoprecipitation from fractionated extracts the hypotonic buffer was modified by

366

adding 100mM NaCl and 0.1% NP40. For immunocomplex formation, protein A/G-coated

367

magnetic beads (Invitrogen) were washed 3 times with the extraction buffer before coating

368

with the primary antibody for 2 hr at 4 ºC in a rotating wheel, followed by 2 washes with the

369

same buffer to eliminates unbound antibody and then extracts are added O/N at 4ºC in the

370

rotating wheel. Immunocomplexes were washed twice and used for western blotting.

371

Western blotting

372

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bioRxiv preprint

Proteins from lysed cells or immunoprecipitates were denatured and loaded on sodium

373

dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene difluoride

374

membranes (Bio-Rad). After blocking with 5% (w/v) BSA or milk the membrane was

375

incubated with the corresponding primary and secondary antibodies (Bio-Rad). The specific

376

bands were analyzed using Thyphoon or ChemiDoc Imaging Systems (Bio-Rad).

377

Immunoflorescence

378

Cells in cover slips were washed three times and fixed with 4% paraformaldehyde in PBS

379

[pH 7.4] for 10min; washed again; permeabilized (PBS [pH7.4], 0.5% Triton X-100, 0.2%

380

BSA) for 5min; blocked (PBS [pH7,4, 0,05% Triton X-100, 5% BSA) for 1h at room

381

temperature; incubated with primary antibody over night at 4ºC, washed three times for 5

382

min and incubated with the secondary antibody for 1h at room temperature. Slides were

383

mounted and images were acquired using a SP5 confocal microscope (Leica) with a 63x

384

objective. Fluorescence intensity was quantified using Image J software. For each

385

experiment, 3 different fields were evaluated per slide.

386

Cell-growth curves

387

Cell growth was determined by colorimetry. For that, cells were seeded at a density of 20,000

388

cells per well in a Corning 12-well plate and treated as indicated for 1 to 6 days. After that

389

cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

390

(MTT) (Sigma-

Aldrich) in a 1:10 ratio in a culture medium and incubated at 37 °C for 3 h.

391

In living cells, MTT is reduced to formazan, which has a purple color. The medium was

392

removed, and the formazan was resuspended in dimethylsulfoxide (DMSO), transferred to

393

p96 plates and analyzed by a Spectra FLUOR (Tecan) at 542 nm. Viability was measured in

394

four independent experiments and then duplicated.

395

RT-qPCR

396

Total RNA was extracted from 3 replicates of colorectal cells using TRIzol reagent

397

(Invitrogen). Reverse transcription of 1

g of RNA was performed according to the

398

manufacturer’s instructions Reagents and detection systems were from Applied biosystems.

399

18S ribosomal RNA primers served as a nonregulated control. Relative expression was

400

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bioRxiv preprint

calculated using the Ct method, expressed as 2

−ΔΔ

[63]

. The PCR efficiency was

401

approximately

100%.

402

Human samples

403

Patient samples were derived from surgical removal at (i) Fundación Jimenez Diaz

404

University Hospital, General and Digestive Tract Surgery Department: 95 patient samples

405

diagnosed with stage II CRC; (ii) at Hospital Clínico San Carlos (HUCSC) 56 primary

406

samples diagnosed with stage IV CRC, and 54 paired liver metastases. The Institutional

407

Review Board (IRB) of the Fundación Jimenez Diaz Hospital, reviewed and approved the

408

study, granting approval on December 9, 2014, by the act number 17/14. All patients gave

409

written informed consent for the use of their biological samples for research purposes.

410

Fundamental ethical principles and rights promoted by Spain (LOPD 15/1999) and the

411

European Union EU (2000/C364/01) were followed. In addition, all patients’ data were

412

processed according to the Declaration of Helsinki (last revision 2013) and Spanish National

413

Biomedical Research Law (14/2007, of July 3).

414

TMA, immunohistochemistry, and quantification.

415

Tissue microarrays (TMA) with stage II and stage IV (primary tumors and their paired liver

416

metastases) containing 95, 56 and 54 cores respectively, were constructed using the MTA-1

417

tissue arrayer (Beecher Instruments, Sun Prairie) for immunohistochemistry analysis. Each

418

core (diameter 0.6 mm) was punched from pre-selected tumor regions in paraffin-embedded

419

tissues. We chose central areas from the tumor, avoiding foci of necrosis. Staining was

420

conducted in 2-

m sections. Slides were deparaffinized by incubation at 60

C for 10 min and

421

incubated with PT-Link (Dako, Agilent) for 20 min at 95

C in low pH to detect SIRT1, or

422

high pH to detect

-Catenin antigens. To block endogenous peroxidase, holders were

423

incubated with peroxidase blocking reagent (Dako, Agilent) and then with the following

424

dilutions of antibodies: anti-SIRT1 (1:50) overnight at 4

C, and anti-

-Catenin (1:500) for

425

20 min at room temperature. All previously described antibodies presented high specificity.

426

After that, slides were incubated for 20 min with the appropriate anti-Ig horseradish

427

peroxidase-conjugated polymer (EnVision, Dako, Agilent). Sections were then visualized

428

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bioRxiv preprint

with 3,3’-diaminobenzidine (Dako, Agilent) as a chromogen for SIRT1 and

-catenin or with

429

the chromogen HRP Magenta (Dako, Agilent) for AceH3K9, for 5 min and counterstained

430

with Harrys’ Haematoxylin (Sigma Aldrich, Merck). Photographs were taken with a stereo

431

microscope (Leica DMi1). According to the human protein atlas (available at

432

http://www.proteinatlas.org), a human testis tissue was used as a positive control for anti-

433

SIRT1. Immunoreactivity was quantified blind with an Histoscore (H score) that considers

434

both the intensity and percentage of cells stained for each intensity (low, medium, or high)

435

following this algorithm (range 0–300): H score = (low%) × 1 + (medium%) × 2 + (high %)

436

× 3. Quantification for each patient biopsy was calculated blindly by 2 investigators (MJFA

437

and JMU). AceH3K9 and SIRT1 showed nuclear staining, whereas

-catenin was in the

438

nucleus and cytoplasm. Clinicopathological characteristics of patients are summarized in

439

Table 2.

440

Statistical analyses

441

To determine whether each of the antigens evaluated here were well-modelled by a

442

normal distribution, Kolmogorov-Smirnov test was used. Comparisons between two

443

independent groups (stage II tumors versus stage IV tumors) were performed with

444

non-parametric Mann-Whitney U-test. Correlations with parametric variables were

445

performed with Pearson test (P) and with non-parametric variables with Spearman

446

test (S). Association analysis between SIRT1 protein levels and cytoplasmic

447

−

catenin levels was performed with Chi-square test, considering median of

448

cytoplasmic

−

catenin Hscore or SIRT1 expression levels as cut-off point to separate

449

samples between high- or low- expression levels.

450

Analysis between two sample groups were performed with Student’s t test, and for

451

multiple comparisons ANOVA with Bonferroni’s post-test was used. All statistical

452

analyses were performed with SPSS IBM software v.24. P-values<0.05 were

453

considered statistically significant.

454

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

Ethics

455

Patient samples were derived from surgical removal at Fundación Jimenez Diaz

456

University Hospital, General and Digestive Tract Surgery Department: 95 patient

457

samples diagnosed with stage II CRC. The Institutional Review Board (IRB) of the

458

Fundación Jimenez Diaz Hospital, reviewed and approved the study, granting

459

approval on December 9, 2014, act number 17/14.

460

Colon cancer tissues were collected using protocols approved by the corresponding

461

Ethics Committees and following the legislation. All patients gave written informed

462

consent for the use of their biological samples for research purposes. Fundamental

463

ethical principles and rights promoted by Spain (LOPD 15/1999) and the European

464

Union EU (2000/C364/01) were followed. All patients' data were processed

465

according to the Declaration of Helsinki (last revision 2013) and Spanish National

466

Biomedical Research Law (14/2007, July 3).

467

468

Acknowledgements

469

We thank Sergio Ruiz Reyes for excellent technical assistance.

470

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constitutive Wnt signaling. Proc Natl Acad Sci U S A. 2010;107: 9216–9221.

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pluripotent cells by regulating Wnt signaling. Cell Biosci. 2015;5.

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2017;474: 1321–1332. doi:10.1042/BCJ20170042

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56. García-Martínez JMM, Chocarro-Calvo A, Moya CMM, García-Jiménez C.

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cells. Diabetologia. 2009;52: 1913–1924. doi:10.1007/s00125-009-1429-1

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Martinez JM, Castaño A, De La Vieja A. From obesity to diabetes and

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cancer: Epidemiological links and role of therapies. Br J Cancer. 2016;114:

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716–722. doi:10.1038/bjc.2016.37

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signalling by microbiota and immunity in diabetes. Endocr Relat Cancer.

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glucose drives selective EP300 activity in colorectal cancer. Locasale JW,

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treat colorectal cancer: Strategies to improve current therapies (Review). In:

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International Journal of Oncology [Internet]. 2023 [cited 10 Nov 2023] pp.

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24–62. doi:https://doi.org/10.3892/ijo.2022.5472

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in Cancer Development and an Overview of Therapeutic Options. Cells.

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2023;12. doi:10.3390/CELLS12070990

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63. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using

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real-time quantitative PCR and. Methods. 2001;25: 402–408.

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doi:10.1006/meth.2001.1262

680

681

Figure legends

682

Figure 1.

1,25(OH)

Reverses SIRT1 Inactivation by Wnt through

−

catenin

683

relocation

684

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

(A)-(G) HCT 116 or HT-29 CRC cells cultured under standard conditions and where

685

indicated, treated with Wnt3A (100 ng/ml) or LiCl (40 mM, to mimic Wnt3A) and/or

686

,25-dihydroxyvitamin D3 (1,25(OH)2D3),100 nM, added 24 hours before

687

harvesting. Cell extracts were fractionated and nuclear extracts (NE) of indicated

688

cells were analyzed (A), (C-D) and (G).

689

(A) Western-blot analysis of the effects of Wnt3A, LiCl and/or 1,25(OH)2D3 on the

690

acetylation levels of SIRT1. Nuclear extracts (NE) from HCT 116 CRC cells were

691

immunoprecipitated (IP) using anti-acetyl-lysine antibodies. Representative blots

692

and statistical analysis using TBP in the Flow though (FT) as loading controls.

693

(B) Confocal imaging of the effect of 1,25 (OH)2D3 on SIRT1 levels (green) in HCT

694

116 CRC cells cultured in the presence/absence of Wnt3A proteins.

695

effects on the SIRT1 /

−

catenin interaction in the nuclei of CRC

696

HT-29 cells. Input (5%) and flow through (FT) are shown. ERK2 serves as loading

697

control.

698

(D) Western-blot analysis of the effects of Wnt3A, LiCl and/or 1,25(OH)2D3 on the

699

acetylation levels of nuclear

−

catenin. Nuclear extracts (NE) from the indicated

700

CRC cells were immunoprecipitated using anti-acetyl-lysine antibodies.

701

Representative blots and statistical analysis using TBP in the Flow though (FT) as

702

loading controls.

703

(E) Confocal imaging analysis of 1,25 (OH)

effects on nuclear exclusion of

704

−

catenin in HCT 116 cells; nuclear envelope, marked with Lamin B antibody (red)

705

and

−

catenin (green). Right panel: Fluorescence intensity was quantified in 3

706

independent experiments using ImageJ software and for each experiment, 3 different

707

fields were evaluated per slide. Scale bars: 25µm.

708

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

(F)-(G) analysis of the effect of 1,25 (OH)2D3 on the expression of Wnt-target genes

709

in CRC cells HT-29. (F) RT-qPCR analysis of cyclin D1 (CRCND1) and MYC.

710

Values normalized with endogenous control (18S RNA) are referred as fold induction

711

over cells without 1,25(OH)2D3. (G) western-blot analysis of cyclin D1 protein

712

levels. Representative blots and statistical analysis using TBP as loading control.

713

Statistical analysis by Student t-test of 3 independent experiments; values represent

714

mean ± SEM of triplicates; * p< 0.05; ** p< 0.01; *** p< 0.001.

715

Figure 2

SIRT1 Activity Decline and Nuclear

-catenin Rise in Human CRC

716

Progression

717

Analysis of SIRT1 levels and activity in TMAs of human CRC samples. Box plots

718

for: SIRT1expression (A) and AceH3K9 expression (B), as percentage of positive

719

stained cells, and cytoplasmic

-catenin Hscore expression (C). (D) Bar-graph

720

showing the percentage of CRC human samples with

-catenin nuclear pattern.

721

P<0.05 were considered statistically significant. (E) Association analysis between

722

SIRT1and cytoplasmic protein levels of

-catenin in all CRC samples (primary

723

tumours of stage II & stage IV primary and liver-paired metastases) as described in

724

the methods section under the paragraph

human samples

. Association analysis was

725

performed with Chi-square test.

726

(F) Correlation analysis between cytoplasmic

-catenin and SIRT1 protein levels on

727

CRC patient biopsies of stage II & stage IV primary tumours and their liver-paired

728

metastases. Pearson (P) or Spearman (S) analyses.

729

(G) Representative micrographs at 40X from immunostaining for SIRT1 and its

730

targeted substrates AceH3K9 and

-catenin from the same CRC patient. Scale bars:

731

20µm.

732

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

(H) CRC patient samples with high SIRT1 levels show high levels of AceH3K9 and

733

mainly nuclear

-catenin. Representative micrographs at 40X; scale bars: 20µm.

734

Figure 3

SIRT 1 is required for nuclear exclusion of

−

catenin by 1,25(OH)

735

HCT 116 CRC cells were cultured with the indicated sirtuin inhibitors or activators

736

and treated or not with 1,25 (OH)

as previously indicated. NAA: Nicotinamide

737

(300 µM), general sirtuin inhibitor. EX527: Selisistat (10

M), selective SIRT1

738

inhibitor. SRT1720 (10

M): selective SIRT1 activator. The effects were analyzed by

739

confocal imaging, scale bars: 25µm. (A), (G); western blotting of nuclear extracts

740

using TBP as loading control (B-D), (H) or proliferation curves (E-F), (I).

741

(A)-(B) Effects of 1,25 (OH)

and NAA on

-catenin subcellular localization. (A)

742

Confocal imaging with nuclear envelope marked with Lamin B antibody (red) and

743

-catenin (green). (B) Western-blot analysis. Representative blots and statistical

744

analysis. (C) Effect of EX527 on nuclear levels of

-catenin, analysed as in (B). (D)

745

Effect of SIRT1 depletion using a mix of siRNAs from Dharmacon on nuclear levels

746

-catenin.

747

(E-F) Effect of EX527 on the 1,25(OH)

-driven blockade of HCT 116 CRC cell

748

proliferation. (G) Effect of the SIRT1 activator SRT1720 on subcellular localization

749

-catenin analysed by confocal imaging with nuclear envelope marked in red with

750

Lamin B antibody and

-catenin (green). (H) Dose-dependent effect of the specific

751

SIRT1 inducer SRT1720 on nuclear

-catenin levels. (I) Proliferation curves to

752

compare the response to 1,25 (OH)

and SRT1720 in CRC cells.

753

Statistical analysis by Student t-test (B-F) and (I) or ANOVA (H) of 3 independent

754

experiments; values represent mean ± SEM of triplicates; * p< 0.05; ** p< 0.01; ***

755

p< 0.001.

756

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

Figure 4

Constitutively active SIRT1 mutant exclude

−

catenin from the nucleus

757

as 1,25(OH)

758

HCT 116 CRC cells cultured in DMEM were transiently transfected with pcDNA

759

expression vectors: empty (-), myc tagged SIRT1 wild type (WT) or mutants: H363Y

760

inactive or K610R, 48 h before harvesting. LiCl was used at 40 mM and 1,25

761

(OH)

(100 nM) was added where indicated for the last 24 h. Extracts were

762

fractionated. (A-C) Western blot analysis of nuclear extracts (NE) using TBP as

763

loading control and MYC as expression control; representative western-blots and

764

statistical analysis. (D) Confocal imaging, scale bars represent 25

765

(A) Effect of SIRT1 expression, wild type (WT) or indicated activity SIRT1 mutants

766

(inactive H363Y or active K610R) on the acetylation levels of nuclear

-catenin

767

analysed after immunoprecipitation (IP) with anti-acetyl lysine antibodies; FT: Flow

768

through.

769

(B) Effect of SIRT1 expression (WT and mutants) on nuclear levels of

-catenin.

770

of the effect of SIRT1 expression (WT and

771

mutants) on nuclear

-catenin levels.

772

(D) Confocal imaging of the myc-tagged SIRT1 mutant (red),

-catenin (green),

773

Lamin B to mark the nuclear envelope (white). Merge panels at the bottom.

774

Statistical analysis by One Way ANOVA of n=3 independent experiments. Values

775

represent mean ± SEM; *p<0.05; **p<0.01; *** p<0.001.

776

Figure 5

SIRT1 activity offers a surrogate target in vitamin D unresponsive and

777

SIRT1 expressing CRC.

778

HCT 116 derived cells were obtained by stable knock-down of Vitamin D receptor

779

(VDR) using specific shRNA targeting VDR to obtain Sh VDR cells, that were

780

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

compared to ShControl cells that contain normal VDR levels [19]. Cells were

781

cultured in DMEM under standard conditions, treated with 1,25 (OH)

and

782

fractionated as previously described; nuclear extracts were analyzed. For Panel C

783

cells were transfected to express SIRT1 mutants H363Y and K610R to comparable

784

levels or the empty vector (

785

(A) Western blot analysis of nuclear

-catenin response to 1,25 (OH)

in cells

786

previously stimulated with LiCl and depleted or not of VDR.

787

(B) Effect of 1,25 (OH)

-catenin subcellular localization in cells previously

788

stimulated with LiCl. Lamin B (red) marks the nuclear envelop to show nuclear

789

accumulation or exclusion of

-catenin (green) in response to 1,25 (OH)

. Scale

790

bars represent 25 μm.

791

-catenin response to 1,25 (OH)

in Sh VDR cells compared to Sh

792

Control cells, upon expression of the SIRT1 activity mutants H363Y or K610R.

793

Levels of expression of mutants are detected with anti-MYC antibody. VDR levels

794

are shown for depletion control. TBP as loading control.

795

(D) Nuclear

-catenin responses to 1,25 (OH)

or to the SIRT1 activator SRT1720

796

in Sh VDR and Sh Control cells. Representative western blots and statistical analysis.

797

TBP as loading control and VDR levels as depletion control.

798

(E) Confocal imaging of the nuclear

-catenin (green) response to 1,25 (OH)

799

the SIRT1 activator SRT1720 in Sh Control and Sh VDR cells. Lamin B at the

800

nuclear envelop is shown in red.

Scale bars represent 25 μm.

801

(F) Nuclear levels of MYC AND Cyclin D1 in response to 1,25 (OH)

or to the

802

SIRT1 activator SRT1720 in Sh VDR and Sh Control cells. Representative western

803

blots and statistical analysis. TBP as loading control.

804

(G) Scheme representative of the new 1,25 (OH)

driven mechanism to block

805

cancer cell proliferation through nuclear exclusion of

-catenin

806

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this version posted January 24, 2024.

bioRxiv preprint

Statistical analysis by One-Way ANOVA of 3 independent experiments; values

807

represent mean ± SEM of triplicates; * p< 0.05; ** p< 0.01; *** p< 0.001.

808

Supporting information

809

Supplementary Figure S1: Vitamin D re locates

-catenin oppositely to Wnt and

810

glucose and independently of whether is added before or after in CRC cells.

811

HCT 116 or HT-29 colon cancer cells were cultured in DMEM and treated with

812

Wnt3A (100 ng/ml) or LiCl (40nM) where indicated, before addition of

813

1,25(OH)

, 100 nM for the last 24 h. Extracts were fractionated, and nuclear

814

extracts (NE) were analyzed.

815

(S1A) Western-blot analysis of 1,25 (OH)

effects on nuclear

-catenin levels in

816

CRC cells treated as indicated. TBP as loading control. Representative blots and

817

statistical analysis.

818

(S1B) Confocal imaging of nuclear

-catenin accumulation by Wnt3A in HCT 116

819

cells and its reversal by 1,25(OH)

representative photographies. Scale bars:

820

25µm. Right panel: fluorescence intensity was quantified in 3 independent

821

experiments using ImageJ software and for each experiment, 3 different fields were

822

evaluated per slide.

823

(S1C) RT-qPCR of CYCLIN D1 (CCND1) and MYC from HCT 116 cells. Values

824

normalized with endogenous control (18S) are referred as fold induction over cells

825

without 1,25 (OH)

826

(S1D) Order of addition analysis of the effects of 1,25 (OH)

on nuclear exclusion

827

-catenin in CRC cells analyzed by western-blotting. Lamin A/C were used as

828

loading controls. Left: cells were treated with 1,25 (OH)

for 24 h and then treated

829

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

or not LiCl to mimic Wnt signalling. Right: cells were first treated with LiCl and then

830

with1,25 (OH)

831

Statistical analysis of 3 independent experiments by One-Way ANOVA in (A) or

832

Student t-test (B) to (D)

; values represent mean ± SEM of triplicates; * p< 0.05; **

833

p< 0.01; *** p< 0.001.

834

Supplementary Figure S2: CRC cell lines respond similarly to SIRT inhibition

835

with NAA, or SIRT1 depletion using several SIRT1 specific siRNAs.

836

Cells were cultured and treated as in previous figures.

837

(S2A)

Western-blot analysis of the effects of 1,25 (OH)

and the sirtuin inhibitor

838

nicotinamide (NAA) on

-catenin nuclear levels; TBP as loading control.

839

Representative blots and statistical analysis.

840

(S2B) Comparison of SIRT1 depletion using an siRNA from Santa Cruz

841

Biotechnology and a mixture of siRNAs from Dharmacon. Representative western

842

blots. TBB serves as loading control.

843

(S2C) Western-blot analysis of the effect of SIRT1 depletion by siRNAs on nuclear

844

-catenin levels in HT-29 CRC cells. Representative picture and statistical analysis.

845

Statistical analysis by One-Way ANOVA of 3 independent experiments; values

846

represent mean ± SEM of

triplicates; * p< 0.05; ** p< 0.01; *** p< 0.001.

847

Author contributions

848

José Manuel García-Martínez, conceptualization, data curation, formal analysis,

849

validation, investigation, methodology; Ana Chocarro-Calvo, resources, formal

850

analysis, supervision, investigation; Javier Martínez-Useros, formal analysis,

851

investigation, methodology; Nerea Regueira-Acebedo: methodology and formal

852

The copyright holder for this

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bioRxiv preprint

García-Martínez et al. Figure 1

-catenin

SIRT1 (IP) /ERK2

(fold induction)

TBP

SIRT1

1,25(OH)

-catenin

NE HT-29

Nuclear

-catenin

(fold induction)

1,25(OH)

LiCl

1,25(OH)

catenin

Lamin B

LiCl

***

SIRT1 (IP) / TBP

(fold induction)

TBP

SIRT1

NE HCT116

***

IP Acetyl Lysine

***

5
4

1,25(OH)

Wnt3a LiCl

-catenin (IP) / TBP

(fold induction)

TBP

-catenin

NE HCT116

***

IP Acetyl
Lysine

***

1,25(OH)

Wnt3a LiCl

0.5

1.5

Wnt3A

1,25(OH)

SIRT1

HCT116

1,25(OH)

RNA

(fold induction)

***

0.5

MYC

CCND1

***

HT-29

TBP

Cyclin D1

NE HT-29

***

0.5

1,25(OH)

Cyclin D1 / TBP

(fold induction)

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

***

TBP

-catenin

1,25(OH)

NAA

β-catenin/TBP

(fold induction)

-catenin

TBP

***

0.4

0.8

1.2

SRT1720 (

García-Martínez et al. Figure 3

1,25(OH)

-catenin

Lamin B

NAA

β-catenin / TBP

(fold induction)

***

0.5

1.5

EX527

TBP

-catenin

β-catenin / TBP

(fold induction)

0.5

1.5

1,25(OH)

SIRT1 siRNA

SIRT1

-catenin

TBP

1,25(OH)

Vehicle

Cell proliferation

(fold induction)

Days 0

1,25(OH)

Vehicle

1,25(OH)

-catenin

Lamin B

SRT1720

1,25(OH)

Vehicle

SRT1720

Cell proliferation

(fold induction)

***

Days 0

-EX527

+EX527

***

1,25(OH)

β-catenin / TBP

(fold induction)

0.5

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

-catenin

SIRT1-MYC

K610R

Merge

LaminB

H363Y

TBP

-catenin

(IP)

TBP

(fold induction)

IP Acetyl Lysine

0.5

1.5

MYC

SIRT1-MYC

García-Martínez et al. Figure 4

***

β-catenin/TBP

(fold induction)

***

0.5

1.5

MYC

-catenin

TBP

SIRT1-MYC

***

TBP

-catenin

-catenin/TBP

(fold induction)

SIRT1

MYC

SIRT1-MYC

WT K610R H363Y

1,25(OH)

0.5

1.5

***

The copyright holder for this

this version posted January 24, 2024.

bioRxiv preprint

García-Martínez

et al. Figure 5

1,25(OH)

-catenin

Lamin B

Sh Control

Sh VDR

-catenin / TBP

(fold induction)

***

0.5

1.5

1,25(OH)

Control

VDR

-catenin

β-catenin / TBP

(fold induction)

1,25(OH)

***

Sh Control

Sh VDR

0.5

1.5

-catenin

VDR

MYC

SIRT1-MYC

R
H

***

Sh VDR

SIRT1720

Sh Control

1,25(OH)

0.5

1.5

Sh Control

Sh VDR

1,25(OH)

SRT1720

Nuclear

-catenin

(fold induction)

***

-catenin

Lamin B

β-catenin / TBP

(fold induction)

1,25(OH)

SRT1720

***

-catenin

VDR

0.5

1.5

Sh Control

Sh VDR

MYC / TBP

(fold induction)

1,25(OH)

SRT1720

MYC

0.5

1.5

Sh Control

Sh VDR

***

Cyclin D1

Sh Control

Sh VDR

Cycllin D1 / TBP

(fold induction)

SIR

�

FER
ATI

ß-c

aten

FER
ATI

Vit

SIR

ß-c

aten

CYT

ASM

Wnt/

-catenin

targe

t gene

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https://doi.org/10.1101/2024.01.21.576539

doi:

bioRxiv preprint

García-Martínez et al. Figure S1

-catenin

-catenin/TBP

(fold induction)

***

1,25(OH)

***

LiCl

Wnt3A

Lamin B

-catenin

1,25(OH)

-catenin /

Lamin A/C

(fold induction)

0.5

1.5

1,25(OH)

LiCl

0.5

1.5

Lamin A/C

-catenin

Pre-treatment

Post-treatment

+/-

1,25(OH)

LiCl

+/-

1,25(OH)

LiCl

24h

48 h

NE HCT116

24h

48 h

Nuclear

-catenin

(fold induction)

***

NE HCT116

LiCl

Wnt3A

1,25(OH)

Wnt3A

HCT116

1,25(OH)

RNA

(fold induction)

0.5

MYC

CCND1

HCT116

HT-29

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https://doi.org/10.1101/2024.01.21.576539

doi:

bioRxiv preprint

0.5

1.5

García-Martínez et al. Figure S2

β-catenin/TBP

(fold induction)

NE HT-29

1,25(OH)

NAA

-catenin

0.5

1.5

***

β-catenin/TBP

(fold induction)

NE HT-29

SIRT1 siRNA

SIRT1

-catenin

1,25(OH)

SIRT1

SIRT1 siRNA

HCT116

HT-29

HCT116

HT-29

SantaCruz

Dharmacon

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