cancers Article Tetraploidy-Associated Genetic Heterogeneity Confers Chemo-Radiotherapy Resistance to Colorectal Cancer Cells Claudia Galofré 1 , Öykü Gönül Geyik 2,3,† , Elena Asensio 1 , Darawalee Wangsa 2 , Daniela Hirsch 2 , Carolina Parra 1 , Jordi Saez 4 , Meritxell Mollà 4 , Zeynep Yüce 3 , Antoni Castells 1 , Thomas Ried 2, * and Jordi Camps 1,5, * 1 Gastrointestinal and Pancreatic Oncology Team, Institut D’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), 08036 Barcelona, Spain; claudiagalofre@gmail.com (C.G.); neko.nyaa84@gmail.com (E.A.); carolina12pg@gmail.com (C.P.); CASTELLS@clinic.cat (A.C.) 2 Section for Cancer Genomics, Genetics Branch, National Cancer Institute, National Institute of Health, Bethesda, MD 20817, USA; oykugonul89@gmail.com (Ö.G.G.); wangsada@mail.nih.gov (D.W.); daniela.hirsch@nih.gov (D.H.) 3 Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University, 35330 Izmir, Turkey; zeynyuce@gmail.com 4 Radiation Oncology Department, Hospital Clínic de Barcelona, 08036 Barcelona, Spain; josaez@clinic.cat (J.S.); MOLLA@clinic.cat (M.M.) 5 Unitat de Biologia Cel·lular i Genètica Mèdica, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain * Correspondence: riedt@mail.nih.gov (T.R.); JCAMPS@clinic.cat (J.C.) † Current affiliation for Ö.G.G.: Department of Nutrition and Dietetics, Faculty of Health Sciences, İstinye University, 34010 Istanbul, Turkey.  Received: 18 March 2020; Accepted: 27 April 2020; Published: 30 April 2020  Abstract: Tetraploidy, or whole-genome duplication, is a common phenomenon in cancer and preludes chromosome instability, which strongly correlates with disease progression, metastasis, and treatment failure. Therefore, it is reasonable to hypothesize that tetraploidization confers multidrug resistance. Nevertheless, the contribution of whole-genome duplication to chemo-radiotherapy resistance remains unclear. Here, using isogenic diploid and near-tetraploid clones from three colorectal cancer cell lines and one non-transformed human epithelial cell line, we show a consistent growth impairment but a divergent tumorigenic potential of near-tetraploid cells. Next, we assessed the effects of first-line chemotherapeutic drugs, other commonly used agents and ionizing radiation, and found that whole-genome duplication promoted increased chemotherapy resistance and also conferred protection against irradiation. When testing the activation of apoptosis, we observed that tetraploid cells were less prone to caspase 3 activation after treatment with first-line chemotherapeutic agents. Furthermore, we found that pre-treatment with ataxia telangiectasia and Rad3 related (ATR) inhibitors, which targets response to replication stress, significantly enhanced the sensitivity of tetraploid cells to first-line chemotherapeutic agents as well as to ionizing radiation. Our findings provide further insight into how tetraploidy results in greater levels of tolerance to chemo-radiotherapeutic agents and, moreover, we show that ATR inhibitors can sensitize near-tetraploid cells to commonly used chemo-radiotherapy regimens. Keywords: tetraploidy; whole-genome duplication; chemotherapy; radiotherapy; drug resistance; colorectal cancer Cancers 2020, 12, 1118; doi:10.3390/cancers12051118 www.mdpi.com/journal/cancers Cancers 2020, 12, 1118 2 of 15 1. Introduction Tetraploidy, the state of having four sets of each chromosome as a consequence of whole-genome duplication (WGD), is a common feature of cancer cells [1]. Several lines of evidence indicate that WGD facilitates the acquisition of a transformed phenotype in human cancers [2], by creating an intermediate state that results in highly aneuploid unstable karyotypes [3]. A seminal study to prove the role of tetraploidy in tumorigenesis showed that p53-null tetraploid cells were able to generate tumors when injected into nude mice, whereas the diploid counterparts did not [4]. In fact, tumors that had undergone genome doubling display a higher tolerance to genomic instability resulting in tumor progression [5]. Additionally, WGD was suggested to act as a driver event that arises early in some cancers [6] and was shown to predict worse overall survival in different cancer types, independent of established clinical prognostic factors [7]. It is well-known that tetraploidy can be induced through different mechanisms, including cytokinesis failure, endoreduplication, mitotic slippage and, less commonly, cell fusion [8]. Recently, environmental stress such as hyperthermia was shown to cause polyploidization in several cancer cell lines [9]. In principle, newly generated tetraploid cells are more likely to result in highly aneuploid karyotypes across consecutive cell divisions due to increased rates of chromosome mis-segregation, which might hamper cellular growth [10]. However, tumor cells can alleviate the impaired fitness. For example, inactivation of the Hippo pathway, either by depletion of LATS2 or by expression of a constitutively active version of YAP, is sufficient to restore proliferative capacity of tetraploid cells [11]. Moreover, other studies suggested that DNA metabolism is compromised in chromosomal unstable cells [12] and near-tetraploid cells [13], which display higher amounts of replication stress than stable and diploid counterparts, respectively. Several mechanisms, by which mild replication stress might cause aneuploidy, have been described, including increased mitotic microtubule growth rates [14] and centrosome disengagement [15]. A direct consequence of tetraploidy is the increase in the levels of chromosome instability (CIN), resulting in highly aneuploid genomes [5,13]. It was suggested that CIN results in tumors with karyotypically heterogeneous subpopulations that will often outcompete other tumor cell populations when exposed to selective pressures [16,17]. Therefore, it is plausible to hypothesize that CIN and tetraploidization confer multidrug resistance, including resistance to some of the most commonly used chemotherapeutic agents, by creating greater levels of genetic diversity and thus promoting the emergence of resistant clones [18–20]. In fact, previous studies already suggested high sensitivity of tetraploid clones to the inhibition of cell cycle or mitotic regulators, including checkpoint kinase 1 [21], aurora kinase B [22], kinesin family member 11 [23], and MPS1 [24]. Nevertheless, there is still an unmet need to identify novel strategies to eradicate polyploid cells by identifying unique features and mechanisms to sensitize these cells to conventional chemo-radiotherapeutic treatments [25]. In the present work, we systematically assessed proliferation rates and levels of resistance to first-line and other commonly used chemotherapeutic agents as well as to ionizing radiation of tetraploid colorectal cancer (CRC) cell lines and non-transformed cells compared to their diploid counterparts. Additionally, we observed that treating tetraploid cells with an ATR inhibitor, which targets response to replication stress, reverted the resistant phenotype and decreased the number of viable cells to those levels observed in their diploid counterparts. This study provides evidence that tetraploidy is associated with multi-drug resistance, and thus novel strategies are needed to identify the Achilles’ heel of tetraploid cells to induce sensitivity to current cancer treatments. 2. Results To assess to what extent tetraploidy confers therapy resistance, we used three CRC cell lines (DLD-1, RKO and SW837), the human non-transformed RPE1 cell line, and their tetraploid counterparts. DLD-1 and RKO are mismatch repair deficient colon cancer cells with pseudodiploid stable karyotypes (2N = 46 for DLD-1 and 2N = 47–50 for RKO). In contrast, SW837 is a mismatch repair proficient rectal cancer cell line with a 2N = 40 [26,27]. Near-diploid and near-tetraploid (hereinafter referred to as 2N Cancers 2020, 12, 1118 3 of 15 and 4N, respectively) Cancers 2020, 12, xclones doi for each of the CRC cell lines were generated in our laboratory 3 of 16and levels of chromosome instability were previously described [13]. (hereinafter referred to as 2N and 4N, respectively) clones for each of the CRC cell lines were generated in our laboratory and levels of chromosome instability were previously described [13]. 2.1. Growth Retardation of Near-Tetraploid Cells in Untreated Conditions and Diverse Tumorigenic Potential 2.1. Growth Retardation of Near-Tetraploid Cells in Untreated Conditions and Diverse Tumorigenic To establish Potential a baseline for cellular growth, we first assessed whether 2N and 4N cells exhibited the same cellular fitness in untreated conditions. Cellular proliferation analyzed by colony formation To establish a baseline for cellular growth, we first assessed whether 2N and 4N cells exhibited assays showed that the same 4N cells cellular expanded fitness in untreatedsignificantly slower conditions. Cellular than 2N analyzed proliferation cells forby allcolony cancer cell lines tested, formation when assessing both the assays showed thattotal number 4N cells expanded ofsignificantly colonies (Figure 1A2N slower than and cellsFigure S1) and for all cancer the tested, cell lines overall area of when assessing both the total number of colonies (Figure 1A and Figure S1) and growth (Figure 1B and Figure S1). Conversely, non-transformed RPE1 4N cells displayed significantly the overall area of growth (Figure 1B and Figure S1). Conversely, non-transformed RPE1 4N cells displayed higher proliferation capacity than 2N cells (Figure 1A,B). Of note, RPE1 cells displayed larger colony significantly higher proliferation capacity than 2N cells (Figure 1A,B). Of note, RPE1 cells displayed areas compared to CRC larger colony cells areas lines due compared tocells to CRC its growing lines due topattern (Figure its growing pattern1B and 1B (Figure Figure S1). S1). and Figure Proliferative Figure 1. Figure and tumorigenic 1. Proliferative and tumorigeniccapacities of 2N capacities of 2Nand and4N4N cells. cells. Quantification Quantification of colony of colony formation assay by (A) number of colonies and (B) area of occupancy in one 2N and clones formation assay by (A) number of colonies and (B) area of occupancy in one 2N and two 4N two 4N clones derived from the different cell lines. Please note that analysis of the number of colonies for the RKO derived from the different cell lines. Please note that analysis of the number of colonies for the RKO cell cell line was not possible due to its dispersed phenotype in the colony-forming assay. Mean of at least line was nottwo possible replicatesdue to three from its dispersed phenotype independent experimentsin with the colony-forming assay. Mean SD is shown. Measurements of at least two of tumor replicates from three independent experiments with SD is shown. Measurements of tumor volumes Cancers 2020, after injecting 12, x; doi: (C–E) CRC and (F) non-transformed RPE1 2N and 4N cellswww.mdpi.com/journal/cancers subcutaneously in athymic nude mice. A.u., arbitrary units. *, p < 0.05; **, p < 0.01; ***, p < 0.001. To analyze their capacity to induce tumorigenesis, 1 × 106 2N and 4N cells from DLD-1, RKO, SW837 and RPE1 cell lines were injected subcutaneously in athymic nude mice. For RKO and SW837, results indicated that 4N cells show the same tumorigenic capacity as 2N cells with no statistical differences in tumor growth. As for DLD-1, 2N and 4N cells displayed the same capacity to form Cancers 2020, 12, 1118 4 of 15 tumors, but 4N cells displayed a growth pattern significantly slower than 2N cells (p = 0.03), which is in line with the in vitro data. In contrast, tetraploidization of RPE1 cells did confer tumorigenic properties compared to the non-tumorigenic 2N RPE1 cells (p < 0.001) (Figure 1B–E). The higher capacity of RPE1 4N cells to induce tumorigenesis in mice agrees with their enhanced clonogenicity. 2.2. Near-Tetraploid Cells Exhibit Tolerance to Treatment with First-Line and Other Chemotherapeutic Agents To explore to what extent tetraploidy provides a selective advantage in therapy resistance, we evaluated the effect of first-line chemotherapeutic agents used in CRC patients, i.e., 5-fluorouracil, oxaliplatin, and FOLFOX. Three CRC cell lines and the non-transformed RPE1 cells were exposed to increasing concentrations of 5-fluorouracil and oxaliplatin emulating clinically used concentrations. Cellular viability was measured at 72 h and each experiment was performed in triplicates. Compared to its corresponding untreated cell culture, 4N clones from all cell lines showed a general multidrug resistant phenotype compared to 2N clones (Figure 2). 4N cells were significantly more resistant than their 2N counterparts to the pyrimidine analog 5-fluorouracil at a dose range from 5 to 100 µM (Figure 2A–C). Additionally, all 4N clones showed significant resistance to oxaliplatin when administrated at a dose range between 2 and 100 µM (Figure 2D–F). Similarly, post-tetraploid RPE1 also displayed increased tolerance to 5-fluorouracil and oxaliplatin to similar levels as those showed by DLD-1, but less sensitive than RKO and SW837 (Figure S2A,B). Treatment with the combination of both drugs, FOLFOX, did only show a slight additive effect in some conditions (Figure 2G–I and Figure S2C), thus suggesting that the addition of oxaliplatin on top of 5-fluorouracil did not further compromised cellular viability. Cancers 2020, 12, x doi 5 of 16 Cellular Figure 2. Figure viability 2. Cellular viabilityresponse response upon upon treatment treatment with first-line with first-line chemotherapeutic chemotherapeutic agents. Dose- agents. response curves for increasing concentrations of 5-fluorouracil (A–C), oxaliplatin Dose-response curves for increasing concentrations of 5-fluorouracil (A–C), oxaliplatin (D–F) and the (D–F) and the combination of both compounds (G–I) in one 2N and two 4N clones of DLD-1, RKO and SW837 CRC combination of both compounds (G–I) in one 2N and two 4N clones of DLD-1, RKO and SW837 CRC cell lines. Each cellular viability was normalized based on its corresponding non-treated counterpart. cell lines. Each cellular Fitted curves forviability wasfrom two replicates normalized based on three independent its corresponding experiments non-treated are plotted. ANOVA counterpart. test with Fitted curves for two post-hoc Tukeyreplicates was performedfrom to three independent test significance. Dataexperiments are reported asaremeans plotted. ± SD.ANOVA n.s., not test with significant; post-hoc Tukey was *,performed p < 0.05; **, pto < 0.01; ***, p < 0.001; ****, test significance. p < 0.0001. Data are reported as means ± SD. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Since both 5-fluorouracil and oxaliplatin interfere with DNA synthesis, we rationalized that 4N cells might display a general resistance to these drugs due to the double amount of DNA. To test whether the increased amount of DNA in 4N clones is ultimately responsible for the tolerance to these agents, we tested 4N clones with twice the amount of drug concentration and compared with 2N clones. Even in these conditions, all 4N clones displayed higher tolerance to 5-fluorouracil (Figure S3A–C) and oxaliplatin (Figure S3D–F) treatment compared to 2N cells in all CRC cell lines. Post- tetraploid non-transformed RPE1 cells also showed a significative resistant phenotype compared to its diploid counterpart after treatment with double amount of FOLFOX (Figure S3G). Cancers 2020, 12, 1118 5 of 15 Since both 5-fluorouracil and oxaliplatin interfere with DNA synthesis, we rationalized that 4N cells might display a general resistance to these drugs due to the double amount of DNA. To test whether the increased amount of DNA in 4N clones is ultimately responsible for the tolerance to these agents, we tested 4N clones with twice the amount of drug concentration and compared with 2N clones. Even in these conditions, all 4N clones displayed higher tolerance to 5-fluorouracil (Figure S3A–C) and oxaliplatin (Figure S3D–F) treatment compared to 2N cells in all CRC cell lines. Post-tetraploid non-transformed RPE1 cells also showed a significative resistant phenotype compared to its diploid counterpart after treatment with double amount of FOLFOX (Figure S3G). To further test whether WGD promoted increased resistance to first-line chemotherapeutic agents used in CRC patients, we assessed the capacity of 2N and 4N cells to form colonies after exposure to these agents. To establish suitable doses of chemotherapy drugs, we first tested 25 µM for 5-fluorouracil and 20 µM for oxaliplatin, but no colonies were formed due to elevated toxicity. Therefore, we reduced the drug concentration to 5 µM of 5-fluorouracil and 5 µM of oxaliplatin. By assessing the area of growth using these conditions, DLD-1, RKO, SW837, and RPE1 4N cells showed higher colony formation capacity than 2N cells (Figure 3A–D). Of note, treatment with FOLFOX showed the most severe cellular viability impairment in DLD-1 cells. No colonies were observed when either 2N or 4N RKOCancers cells2020, were 12, xtreated doi with oxaliplatin, suggesting that these cells are highly impaired 6 of 16to form colonies under this treatment (Figure 3B). In addition, not only the size but also the number of colonies number was higher of colonies in 4N compared was higher to 2N in 4N compared clones to 2N clones after treatment after treatment (Figure S4A–C).(Figure S4A–C). Of note, RKOOfcells note,did not RKO cells did not form discrete colonies due to the high spread capacity, thus making it impossible form discrete colonies due to the high spread capacity, thus making it impossible to assess the readout to assess the readout of this assay. Overall, these results suggest that although 4N cells displayed a of this assay. slower Overall, growth rate these underresults suggest untreated that although conditions, 4N cells WGD promoted displayed increased a slower resistance growth rate to multidrug under untreated treatments.conditions, WGD promoted increased resistance to multidrug treatments. Clonogenic Figure 3.Figure capacity 3. Clonogenic of 2N capacity of 2Nand and4N4Ncells cells after treatment after treatment with with first-line first-line chemotherapeutic chemotherapeutic agents. Graphs showing agents. Graphs the clonogenic showing the clonogeniccapacity capacityin in one 2Nand one 2N andtwo two4N4N clones clones of DLD-1 of DLD-1 (A),(B), (A), RKO RKO (B), SW837 (C) SW837and(C) RPE1and (D) RPE1cells (D) cells treated treated with with 5 µM 5 µM 5-fluorouracil, 55 µM 5-fluorouracil, µMoxaliplatin oxaliplatin or or thethe combination combination of of both compounds. both compounds. Clonogenic Clonogenic capacitycapacity was assessed was assessed by areabyof area of occupancy occupancy overover the untreated the untreated control at control at 14 to 21 days after treatment. Data are reported as means ± SD (n = 6). n.s., not significant; 14 to 21 days after treatment. Data are reported as means ± SD (n = 6). n.s., not significant; *, p < 0.05; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. ***, p < 0.001; ****, p < 0.0001. Next, we interrogated the observed multidrug tolerance of 4N cells using other specific drugs Next, we interrogated commonly the observed used for therapy multidrug in CRC, such tolerance as irinotecan, of 4N paclitaxel andcells using other gemcitabine. specific In this case, drugs commonly used was treatment for administered therapy in to CRC,DLD-1such as irinotecan, 2N and 4N clones andpaclitaxel and gemcitabine. the MTT readout In this was obtained after 72 case, hours. Treatment with both irinotecan and gemcitabine resulted in a larger percentage treatment was administered to DLD-1 2N and 4N clones and the MTT readout was obtained after 72 h.of 4N cells with higher viability. Significance was reached for most of the concentrations tested for irinotecan, while only high concentrations of gemcitabine exhibited statistically significant difference (Figure S5A,B). In contrast, DLD-1 4N cells showed a statistically significant increased sensitivity to paclitaxel compared with 2N counterparts (p < 0.05) (Figure S5C), potentially associated with its effect on microtubule stabilization. Cancers 2020, 12, 1118 6 of 15 Treatment with both irinotecan and gemcitabine resulted in a larger percentage of 4N cells with higher viability. Significance was reached for most of the concentrations tested for irinotecan, while only high concentrations of gemcitabine exhibited statistically significant difference (Figure S5A,B). In contrast, DLD-1 4N cells showed a statistically significant increased sensitivity to paclitaxel compared with 2N counterparts (p < 0.05) (Figure S5C), potentially associated with its effect on microtubule stabilization. 2.3. Apoptosis is Ubiquitously Activated by First-Line Chemotherapy Treatments To investigate whether the impairment in cellular viability was due to an increase in apoptosis or simply as a consequence of a reduction in cellular proliferation, we conducted a caspase 3 activity assay as a surrogate marker of apoptosis. As the activation and cleavage of caspase 3 changes over time, we first tested three different exposure times (i.e., 6, 16 and 24 h) to FOLFOX treatment (25 µM of 5-fluorouracil and 20 µM of oxaliplatin). As shown in Figure S6A, the highest caspase 3 activity was obtained at 24 h of treatment, and thus used as a readout timepoint. Following these conditions, the caspase Cancers32020, activity 12, x doi assay was performed in DLD-1, RKO, SW837 and RPE1 2N and 4N cells. 7 of 16 Results indicated that FOLFOX treatment induced apoptosis in all cell lines. Despite the fact that cells. Results indicated that FOLFOX treatment induced apoptosis in all cell lines. Despite the fact untreated 2N and 4N cells showed no statistically significant differences in apoptosis activity, 4N cells that untreated 2N and 4N cells showed no statistically significant differences in apoptosis activity, showed4N significantly cells showedless caspaseless significantly 3 activation after treatment caspase 3 activation with FOLFOX after treatment with FOLFOX for all for cancer all cancer cell lines tested (Figure cell lines4A–C). However, tested (Figure 4A–C).noHowever, differences between 2N no differences and 4N between clones 2N and were found 4N clones were found in RPE1in cells under FOLFOX RPE1 cellstreatment under FOLFOX (Figure 4D). (Figure treatment To test4D). whether To test caspase 3 is activated whether caspase as a as 3 is activated response a response to either to either 5-fluorouracil or oxaliplatin individually, we incubated 2N and 4N DLD-1 5-fluorouracil or oxaliplatin individually, we incubated 2N and 4N DLD-1 cells with a single drug, cells with a single drug, and confirmed that caspase 3 was already activated as a result of treating cells with individual and confirmed that caspase 3 was already activated as a result of treating cells with individual agents agents (Figure S6B). Altogether these results indicate that the greater resistance of 4N cells to (Figure S6B). Altogether these results indicate that the greater resistance of 4N cells to chemotherapy chemotherapy agents is, at least partially, due to the fact that 4N cells are less prone to activate agents is, at least partially, due to the fact that 4N cells are less prone to activate apoptosis. apoptosis. Caspase Figure 4.Figure 3 activation 4. Caspase assay 3 activation in in assay 2N2Nand and4N cellsupon 4N cells upon treatment treatment withwith FOLFOX. FOLFOX. Quantification Quantification of caspase3of caspase3 activity activity as a surrogate as a surrogate markerofofapoptosis marker apoptosis in in2N 2Nandand4N4Nclones of (A) clones ofDLD-1, (B) RKO, (A) DLD-1, (B) RKO, (C) SW837 and (D) RPE1 cell lines in both non-treated and after treatment (25 µM (C) SW837 and (D) RPE1 cell lines in both non-treated and after treatment (25 µM of 5-fluorouracil and of 5-fluorouracil and 20 µM of oxaliplatin) conditions. For all these analyses, caspase3 activity was calculated as pmol 20 µM of oxaliplatin) conditions. For all these analyses, caspase3 activity was calculated as pmol of of pNA/min·mg of protein at 24 h after treatment. Three independent experiments were performed pNA/min·mg of protein at 24 h after treatment. Three independent experiments were performed for each for each cell line. Data are represented as means ± SD. n.s., not significant; **, p < 0.01; ***, p < 0.001; cell line. ****, Data p