Elevated glucose promotes DNA replication and cancer cell growth through pRB-E2F1

Abstract


Introduction
6 inhibition (Fig. 2B). Similarly, HG-induced increases in DNA replication fork speed and cell 116 growth were also reduced in E2F1 knockdown cells (Fig. 2C, 2D). 117 We note that, compared to controls, E2F1 knocked down alone led to more EdU 118 incorporated cells in the S phase (Fig. 2B) as well as increased DNA replication fork speed (Fig.  119 2C). Since the activator E2F family members (E2F1-3) have been shown to have extensive 120 functional redundancy and overlap, we speculate that other E2F family members may offset the 121 E2F1 knocked down effect, resulting in increased cells in the S phase and DNA synthesis. To 122 test this possibility, we employed a pan-E2F inhibitor HLM006474 that blocks chromatin 123 accessibility of E2F family members in cells (Y. Ma et al., 2008). Strikingly, HLM006474 124 completely abolished HCT116 cells entering into S phase in the presence and absence of HG 125 (Fig. 2E). Similarly, a remarkable DNA replication fork arrest was also detected in HLM006474-126 treated cells (Fig. 2F). To exclude the potential cell type specific effect, we knock-downed E2F1 127 in HCT116 cells (Fig. S2A) and inhibited E2F1 with the inhibitor in H460 (Fig. S2B) and 128 obtained similar HG effect on DNA replication and cell proliferation. Together, these results 129 indicate that E2F1 plays a crucial role in promoting DNA replication and cell proliferation 130 following HG treatment. 131

Elevated glucose induces E2F1-dependent transactivation through pRB phosphorylation 132
To further establish the role of E2F1 in HG-induced DNA synthesis and cell growth, we 133 confirmed its effect on up-regulation of 7 identified DNA replication genes (Table 1, Fig. 1A) by 134 RT-PCR. As shown in Figure 3A, compared to controls, HG significantly increased the mRNA 135 levels of RRM2, CHAF1A, CHAF1B, PCNA, CCNE2, CLSPN and RBM14. E2F1 knockdown,136 however, significantly reduced the HG-induced up-regulation. Importantly, E2F1 knockdown 137 also reduced HG-induced RRM2 and CHAF1A protein levels (Fig. 3B), suggesting functional 7 relevance of the regulation. Furthermore, we showed that treating cells with the E2F1 inhibitor 139 HLM006474 also blocks HG-induced mRNA and protein levels of DNA replication-associated 140 genes ( Fig. 3C and 3D). Consistently, overexpression of E2F1 up-regulated the mRNA and 141 protein levels of the DNA replication genes and reduced HG-induced up-regulation in cells (Fig. 142 S3A and S3B). These results suggest that E2F functions as a transcription factor up-regulating 143 DNA replication genes following HG treatment. 144 Several groups have reported that E2F1 binds to the RRM2 promoter and activates its 145 transcription (Fang et al., 2015;Mazzu et al., 2018;Rasmussen et al., 2016). We thus carried out 146 ChIP analysis to investigate whether elevated glucose affects the ability of E2F1 to bind to DNA. 147 The assay confirmed the binding of E2F1 to the RRM2 promoter ( Fig. 3E) as well as to the 148 PCNA promoter (Fig. 3F). Importantly, exposure of cells to HG significantly enhanced the 149 binding of E2F1 to both promoters. Together, these results suggest that elevated glucose up-150 regulates DNA replication genes through E2F1-dependent transactivation. 151 Interestingly, a previous study has suggested that treating cells with HG leads to pRB 152 hyper-phosphorylation (Annicotte et al., 2009). pRB is the target of phosphorylation by 153 CDK2/4/6 in the G1 phase of the cell cycle. Generally, once hyper-phosphorylated, pRB 154 alleviates repression of E2F, leading to activation of E2F1-dependent transcription. To test the 155 role of pRB phosphorylation in HG-induced E2F transactivation, we treated cells with HG and 156 assay pRB phosphorylation using phosphorSer807/811-specific antibody. As shown in Figure  157 4A, increased pRB phosphorylation was clearly observed following HG treatment in HCT116 158 cells, suggesting pRB hyperphosphorylation potentially contributed to E2F1 activation. 159 Interestingly, perhaps due to higher levels of pRB phosphorylation in the cell, treating U2OS 160 cells with HG didn't further enhance pRB phosphorylation (Fig. 4A). Significantly, compared to 161 HG-treated HCT116 cells, treating U2OS cells with HG also failed to up-regulate DNA 162 replication genes ( Fig. 4B and S4A) and to enhance DNA synthesis (Fig. 4C). To confirm these 163 results, we treated HCT116 cells with the CDK2/4/6 inhibitor PF-3600 (Freeman-Cook et al., 164 2021) and showed inhibition of pRB phosphorylation (Fig. 4E) also blocks HG-induced up-165 regulation of DNA replication genes ( Fig. 4D and S4B). These results suggest that elevated 166 glucose up-regulates DNA replication associated genes through regulating the pRB-E2F 167 pathway. 168

Regulation of intracellular dNTP levels by HG is dependent on the E2F1-RRM2 axis 169
The role of RRM2 as a proto-oncogene has been recently recognized. RRM2 is an 170 essential component in the holoenzyme ribonucleotide reductase (RNR) that is important for 171 reducing the 2' carbon of NDP to produce dNDP, a rate-limiting step in the DNA de novo 172 pathway. Our finding that HG up-regulates both RRM2 mRNA and protein levels prompts us to 173 test its role in regulating intracellular dNTP levels. Using a previously described PCR-based 174 method (Purhonen et al., 2020) the intracellular dATP, dGTP, dCTP and dTTP levels were 175 measured and calculated at 2.86, 1.04, 3.07, and 10.82 pmol/10 6 cells, respectively. Treating 176 cells with HG led to a rapid and robust increase of all four dNTP levels in the cells (Fig. 5A). To 177 establish the role of RNR in the HG-induced upregulation of dNTP, we treated cells with the 178 RNR inhibitor Triapine at two concentrations, 250 or 500 nM, and observed reduced intracellular 179 dATP and dGTP levels under both conditions. The Triapine treatment also reduced dCTP and 180 dTTP, but to a lesser extent, which is consistent with a previous report (Lin et al., 2011). 181 Importantly, inhibition of RNR clearly prevented HG-induced DNA replication fork progression 182 (Fig. 5B). Finally, we verified the role of E2F1 in HG-induced dNTP up-regulation. As shown in 183 Figure 5C, treating cells with the E2F inhibitor 6476 clearly blocks dATP and dGTP up-184 regulation following HG treatment. Together, these results suggest that elevated glucose 185 enhances dNTP levels through activation of the E2F1-RRM2 axis. 186

Inhibition of E2F1-RRM2 axis alleviates high glucose-induced cancer cell growth 187
We next investigated the contribution of the E2F1-RRM2 axis to cancer cell growth 188 following HG treatment. To better mimic the in vivo environment, we established a short-term 189 three dimensional (3D) tumor spheroid culture (Zoetemelk et al., 2019). As shown in Figure 6A, 190 H460 cells formed spheroid-like round or spherical structures in the 3D cultures. Upon HG 191 treatment, increased size of the spheroids was observed, indicating a stronger cell growth 192 compared with non-HG treated spheroids (Fig. 6A). To confirm the cell growth potential, overall 193 cell viability of total spheroids in the 3D cultures was further determined following HG treatment 194 (Fig. 6B). These results provide additional support for the finding that elevated glucose re-directs 195 cells to cell growth. 196 To investigate the role of E2F1 in HG-induced spheroid growth, we employed lentivirus-197 mediated RNAi approach to knockdown E2F1. The results show that compared with scramble 198 controls, HG-induced spheroid growth was reduced upon E2F1 inhibition (Fig. 6A). Importantly, 199 HG-induced cell viability of total spheroids in the 3D cultures was also reduced in E2F1 200 knockdown cells (Fig. 6B). 201 Next, we investigated the role of RRM2 in HG-induced spheroid growth by treating cells 202 with the RNR inhibitor Triapine. As shown in Figure 6C, HG-induced spheroid growth was 203 indeed blocked upon the RNR inhibition. As expected, HG-induced cell viability of total 204 spheroids in the 3D cultures was also blocked by the inhibition (Fig. 6D). Taken together, these 205 results demonstrate elevated glucose promotes cancer cell growth through E2F1-RRM2 206 activation. 207

Discussion 208
Diabetes mellitus and cancer have a tremendous effect on human health worldwide. 209 Although a number of epidemiological studies have highlighted the link between two diseases 210 (Giovannucci et al., 2010b;Gordon-Dseagu et al., 2013;Wang et al., 2020), the molecular 211 mechanism by which hyperglycemia promotes cancer cell growth remains well defined. In this 212 study, we identified E2F1 as the core transcriptional regulator involved in re-directing cells to 213 DNA replication and cell proliferation under elevated glucose conditions. Among HG-induced 214 E2F1 target genes, we show that activation of RRM2 leads to up-regulation of intracellular 215 dNTP levels, which plays a role in DNA synthesis and cancer cell growth. Together, our findings 216 provide a molecular mechanism by which hyperglycemia promotes cancer cell proliferation. 217 E2F transcription factors are downstream effectors of the tumor suppressor pRB and have 218 a pivotal role in controlling cell-cycle progression. E2Fs also participate in cellular processes 219 beyond the cell cycle, including apoptosis, differentiation and development. However, the role of 220 E2Fs in re-directing cancer cells to proliferation following HG has not been well investigated. 221 By examining the transcriptome data of the HG-treated cells, DNA replication emerges as a 222 significant signature. Furthermore, GSEA analysis revealed E2F1 as the core transcription factor, 223 suggesting hyperglycemia potentially re-directs cancer cells into DNA replication through E2F1-224 mediated transcription. Interestingly, consistent with this notion, regulation of DNA replication 225 genes has been reported in fin tissues in the diabetic zebrafish model (Leontovich et al., 2016). 226 Furthermore, inhibition of GLUT1, a key rate-limiting factor for glucose uptake, blocked growth 227 of pRB-positive triple negative breast cancer (TNBC) (Wu et al., 2020). Notably, pathway 228 enrichment analysis of gene expression data in TNBC also suggests that the functionality of the 229 E2F pathway contributed to the process. Together, those results imply a HG-regulated pRB-230 E2F1 axis in cancer cells. Clearly, it will be intriguing to further assess its contribution in pRB-231 positive cancer patients. 232 Emerging evidence has indicated that RRM2, the small subunit of RNR, is an important

Cell cycle analysis 287
Cell cycle analysis was performed using the Click-iT Plus Edu Kit (#C10632, Thermo 288 Scientific) according to the manufacturer's protocol. In brief, 2 million cells post-treatment were 289 harvested and fixed. EdU was then labeled with Alexa Fluor 488 picolyl azide for 30 min at 290 room temperature. After labeling, total DNA content was stained with 20ng/ml PI (#P3566, 291 Invitrogen) for 1 h at room temperature. Samples were then analyzed by flow cytometry on the 292 NovoCyte platform (ACEA). 293

B44) (#BDB347580, Fisher Scientific). Secondary antibodies of Alexa Fluor 555 goat anti-rat 302
IgG (#A21434, Thermo Scientific) and Alexa Fluor 488 F (ab′)2 goat anti−mouse IgG (#A-303 11017, Thermo Scientific) were used to at 1:500 dilution for 2 h at room temperature. Images of 304 well spread DNA fibers were taken using Leica microscope with X 40 oil immersion objective. 305 100-150 well spread DNA fibers were collected for each condition. Double-labeled DNA fiber 306 lengths were measured in Image J (v1.53k). The rate for DNA replication fork was estimated 307 using the conversion of 2.59 kb/μm as described (Jackson & Pombo, 1998). 308

RNA isolation and RT-qPCR 309
RNA was extracted with the TRIzol reagent (#15596018, Invitrogen) and then reverse-310 transcribed with the reverse transcription supermix (#1708841, Bio-Rad) according to the 311 manufactures' protocols. qPCR was performed using the SYBR supermix (#1708882, Bio-Rad) 312 in the Bio-Rad CFX cycler with CFX Maestro software. Primers for qPCR are listed in Table 3. 313 Results of mRNA relative levels were calculated by 2 -ΔΔCt in normalization to the GAPDH and 314 relative to the control samples. All PCR reactions were performed in technical triplicates. 315

ChIP-qPCR 316
Cells were cross-linked in 1% formaldehyde (#F1635, Sigma-Aldrich) for 10 min at 317 room temperature and followed by attenuation of 125 mM glycine. Nuclei were isolated in NP-318 40 cell lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP40, 15 mM EDTA) for 20 min on 319 ice and spun down 5000 rpm for 5 min at 4°C. Nuclei were then lysed in nuclear lysis buffer (50 320 mM Tris pH 8, 150 mM NaCl, 10 mM EDTA, 1% SDS) for 10 min on ice. Lysed nuclei were 321 then subjected to sonication to generate ~300bp chromatin fragments by confirmation on agarose 322 gel. Immunoprecipitation was continued by incubating the sheared chromatin with E2F1 323 antibody (#3742, Cell Signaling) and IgG control (#2729, Cell Signaling) overnight at 4°C. 324 Protein G beads (#10003D, Thermo Scientific) were added the following day and incubated on a 325 rotator for 4h at 4°C. Immunoprecipitated beads were then washed twice in low salt wash buffer 326 were reverse cross-linked in a 65°C water bath overnight. Reverse cross-linked DNA was 332 isolated by PCR purification kit (Qiangen). 333 ChIP-qPCR was performed using the SYBR supermix (Bio-Rad) in the Bio-Rad CFX 334 cycler with CFX Maestro software. Primers for ChIP-qPCR are listed in Table 3. 1% of starting 335 chromatin was used as input and technical triplicates were performed. The ChIP-qPCR data was 336 analyzed with the Percent Input Method including normalization for both IgG levels and input 337 chromatin going into the ChIP. 338

Three-dimensional (3D) cultures 339
Cells were seeded at 2000 cells per well in a 96-well U-bottom plate (#353077, Corning). 340 The 3D cell culture media was a mixture of the media for 2D cell culture supplemented with 341 10% Matrix (#A1413201, Thermo Scientific). On day 6, sphere colonies were firstly observed 342 under microscope and images were taken using Leica microscope with X 5 bright field objective. 343 The length and width for each spheroid were measured afterwards in Image J (v1.53k). The 344 viability for spheroids was determined by CellTiter-Glo ® 3D Cell Viability Assay Kit (#G9681, 345 Promega). Briefly, 100 µl CellTiter-Glo ® 3D Reagent was added into the wells to be determined, 346 followed by shaking for 5 min. After incubation at room temperature for 25 min, the plate was 347 read on the luminometer plate reader (Promega). The luminescence signals were measured and 348 collected for evaluating the 3D cell growth viability. 349

Intracellular dNTPs measurement 350
Intracellular dNTP levels were determined as previously described (Purhonen et al., 351 2020). Briefly, cell pellets were resuspended in 60% methanol (Fisher Scientific) and then 352 incubated at 95°C for 3 min. The supernatant was collected after centrifugation and transferred 353 into the Amicon Ultra-0.5ml centrifugal filter (#UFC500396, Millipore) for centrifugation again. 354 After centrifugation, the flow through was saved and dried using Speed-Vac. The dried pellet 355 was dissolved in 300 µl of sterile water and stored at -80°C. Determination of dNTP levels were 356 performed by following the PCR based assay as previously described (Purhonen et al., 2020). 357

Statistical analysis 358
All the data are represented as mean ± SD. All the statistical tests were done using 359 Graphpad Prism 9. P values were also generated in Graphpad Prism 9, with *P <0.05, **P 360 <0.01, ***P <0.001, ****P <0.0001, ns represents non-significance. Representative bright field images of H460 derived spheroids in the presence of scramble control 573 or shE2F1 for 6 days following HG exposure. Scale bar: 100µm. B. ATP luminescence signals 574 represented for the total spheroid's viability on the plate. C. Representative bright field images 575 of HCT116-derived spheroids in the presence or absence of Triapine for 6 days following HG 576 exposure. D. ATP luminescence signals represented for the total spheroid's viability on the plate. 577 Results are displayed in mean ± SD of three separate experiments.   A. RT-qPCR of top DNA replication genes in control (scramble) or E2F1 knock down H460 cells following HG exposure. B. Western analysis of CHAF1A, RRM2 and E2F1 protein levels from the same treatment as indicated in 3A. C. RT-qPCR of top DNA replication genes in control or HLM006474 treated HCT116. D. Western blot analysis for CHAF1A, RRM2 and E2F1 from the same treatment as indicated in 3C. E and F. Top: schematic representation of the RRM2 (3E) or PCNA (3F) promoters. ChIP-qPCR analysis of E2F1 binding to the RRM2 and PCNA promoters in the HCT116 cells following HG exposure. Results are displayed in mean ± SD for n=3 replicates.

Figure 4
A. Western blot analysis of total pRB, Ser807/Ser811-phosphorylated pRB and RRM2 in HCT116 and U2OS cells following HG exposure. B. RT-qPCR of top DNA replication genes in HCT116 and U2OS cells following HG treatment. C. Cell cycle pro les of EdU incorporation and DNA content in U2OS cells following HG treatment at the indicated time. The right panel indicates the percentage of EdU-labeled cells in the S phase. D. HCT116 cells were treated with the CDK inhibitor PF-3600 and RT-qPCR of top DNA replication genes was performed following HG exposure. E. Western blot analysis of total pRB and Ser807/Ser811-22 phosphorylated pRB from the same treatment as indicated in 4D. Results are displayed in mean ± SD for n=3 replicates Figure 5 A. Intracellular dNTP levels in HCT116 cells treated with or without Triapine and HG as indicated. B. DNA replication fork progression of Triapine-treated HCT116 cells following HG exposure. The right panel summarizes measuring of 100-150 spread DNA bers for each condition. C. HCT116 cells were treated with the E2F inhibitor HLM006474. Intracellular dATP and dGTP levels were determined following HG. Results are displayed in mean ± SD of three separate experiments.

Figure 6
A. Representative bright eld images of H460 derived spheroids in the presence of scramble control or shE2F1 for 6 days following HG exposure. Scale bar: 100µm. B. ATP luminescence signals represented for the total spheroid's viability on the plate. C. Representative bright eld images of HCT116-derived spheroids in the presence or absence of Triapine for 6 days following HG exposure. D. ATP luminescence signals represented for the total spheroid's viability on the plate. Results are displayed in mean ± SD of three separate experiments.

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