Skip to main content
NCBI home page
Autophagy Reports logoLink to Autophagy Reports
. 2026 Feb 13;5(1):2624259. doi: 10.1080/27694127.2026.2624259

TFEB confers resistance against the chemotherapeutic agent CX-5461

Marjorie Rolland a, Benoît Marchand a, Laure Bessy a, Florence McDuff a, Marie-Josée Boucher a,b,✉
PMCID: PMC12915801  PMID: 41717449

ABSTRACT

Identifying mechanisms underlying chemoresistance is essential for improving the efficacy of chemotherapeutic drugs. Previously, we showed that cancer cells respond to gemcitabine by activating protective signals dependent on the master regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB). However, how gemcitabine triggers these protective responses remains elusive. While gemcitabine primarily aims at disrupting DNA replication, it is also suspected to induce nucleolar stress. In this study, we aimed to examine the effect of gemcitabine on nucleolar stress and investigate whether nucleolar stress inducers could trigger TFEB-dependent protective signals. Besides gemcitabine causing nucleolar stress, the anticancer agent CX-5461, primarily designed to induce nucleolar stress, promoted TFEB nuclear accumulation. Interfering with TFEB improved the sensitivity of cancer cells to both CX-5461 and gemcitabine. Our findings suggest that TFEB provides broad protection against the stress caused by chemotherapeutic drugs, representing a promising target for intercepting chemoresistance and improving the efficacy of anticancer agents.

KEYWORDS: Autophagy, chemoresistance, CX-5461, gemcitabine, nucleolar stress, TFEB

Introduction

Owing to intrinsic and acquired drug resistance, the efficacy of anticancer therapies is often compromised. While some mechanisms have been identified, further research is needed to pinpoint key pathways that confer resistance to cancer cells, as this could reveal targets that make cancer cells more vulnerable to the stress imposed by chemotherapeutics[1]. Our recent study indicated that gemcitabine induces autophagy and enhances lysosomal activity in cancer cells, acting as protective mechanisms against the drug[2]. This aligns well with the idea that the autophagy-lysosome pathway offers protective signals during anticancer treatments[3,4]. Nevertheless, the mechanisms by which anticancer therapies promote the autophagy-lysosome pathway remain elusive. From a mechanistic perspective, our findings showed that interfering with the master regulator of autophagy and lysosomal function, namely transcription factor EB (TFEB), reduces lysosomal function in cancer cells and improves their responsiveness to gemcitabine, suggesting that TFEB contributes to drug resistance[2]. However, it remains unclear how gemcitabine, a chemotherapeutic agent designed to block DNA replication and induce cell death in dividing cells, promotes TFEB function[5]. Notably, chemotherapeutic drugs have also been shown to disrupt cell homeostasis by inducing nucleolar stress[6–9]. Nonetheless, to our knowledge, this has not been directly evaluated for gemcitabine treatment.

Nucleoli are membraneless organelles that play critical roles in ribosome production and assembly. Since cancer cells require substantial ribosome biogenesis to support their high demand for protein synthesis, inhibitors targeting rDNA transcription, such as the selective RNA polymerase I (RNA Pol I) inhibitor CX-5461, have been developed as an anticancer strategy[10,11]. CX-5461 demonstrated promising anticancer efficacy in advanced Phase 1 clinical trials and has received Fast-Track designation from the Food and Drug Administration (FDA)[12,13]. Nevertheless, although interfering with RNA Pol I induces nucleolar stress, the cellular response to CX-5461 remains incompletely understood[13,14]. Furthermore, although some have suggested that CX-5461 may escape resistance mechanisms, this remains to be established[12].

In the present study, we aimed to examine the effect of gemcitabine on nucleolar stress and investigate whether the nucleolar stress inducer CX-5461 could trigger TFEB-dependent protective signals. Herein, we demonstrate that cells responded to gemcitabine and CX-5461 by inducing nucleolar stress. Similar to gemcitabine, CX-5461 caused TFEB to accumulate in the nucleus. Reducing TFEB levels using shRNA strategies decreased the clonogenic growth ability of cancer cells and sensitized them to the chemotherapeutic drugs. These findings support a broad role for TFEB in sensing cellular stress, such as nucleolar stress, which diminishes the effectiveness of chemotherapeutic drugs. Targeting TFEB could be a promising and broader approach to overcoming chemoresistance.

Results and discussion

Gemcitabine and CX-5461 triggered nucleolar stress

To test whether gemcitabine causes nucleolar stress, we examined the structure of the nucleoli. Canonical nucleolar stress is characterized by nucleoli rounding up and the relocation of nucleolar proteins, such as nucleolin (NCL) and nucleophosmin (NPM1), from the nucleoli to the nucleoplasm[7,13]. In response to gemcitabine, HeLa cells exhibited rounded nucleoli, with a decrease in NPM1 staining intensity in the nucleoli and an increase in the nucleoplasm (Figure 1(A)). This staining pattern was similar to the effect of actinomycin D, which was used as a nucleolar stress inducer[7]. Rounded nucleoli and nucleoplasm staining were also detected in gemcitabine-incubated HeLa and MIA PaCa-2 cells immunostained for NCL (Figure 1(B,C)). Staining of the nucleoplasm with NCL was even more pronounced when cells were exposed to CX-5461. Of note, similar NCL redistribution to the nucleoplasm was recently reported in p53-proficient U2OS cancer cells treated with CX-5461[15]. These results support the induction of nucleolar stress by gemcitabine and CX-5461, despite these two chemotherapeutic drugs exhibiting distinct nucleolar phenotypes. Nonetheless, nucleolar stress is recognized as heterogeneous, reflected by various changes in nucleoli organization[7,9,13].

Figure 1.

Figure 1.

Open in a new tab

Nucleolar stress and DNA damage upon gemcitabine and CX-5461 treatment.

(A) HeLa cells were incubated for 24 h with DMSO (control), gemcitabine (5 μM) or 4 h with actinomycin D (5 nM) before immunostaining using an anti-NPM1 antibody. HeLa (B) and MIA PaCa-2 (C) cells were incubated for 24 h with DMSO (control), gemcitabine (10 μM) or CX-5461 (2 μM) before immunostaining using an anti-NCL antibody. Nuclei were stained with DAPI. Scale bars, 10 μm. HeLa (D) and MIA PaCa-2 (E) cells were incubated for 48 h with vehicle (-), gemcitabine (10 μM), or the indicated concentration of CX-5461. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. Relative cell growth of HeLa (F) and MIA PaCa-2 (G) cells incubated with vehicle (DMSO, -), or the indicated concentration of gemcitabine or CX-5461 as measured by crystal violet assays. Graphs represent mean ± standard deviation (SD) from 16 biological replicates of two independent experiments (F) or 24–48 biological replicates of three to six independent experiments (G). Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. ***p < 0.001, ****p < 0.0001.

We also investigated whether CX-5461 induces DNA damage, as we previously reported for gemcitabine treatment[2]. Elevated levels of the DNA damage marker H2AX were detected in CX-5461-incubated cells, although to a lesser extent than upon gemcitabine incubation (Figure 1(D,E)). To provide some information about the DNA damage response initiated by CX-5461, we checked the phosphorylation levels of key kinases activated by DNA damage, namely DNA-PKcs, ATM, and CHK2[16]. As shown in Figure 1D, CX-5461 was less effective than gemcitabine in promoting the phosphorylation of DNA-PKcs, and no ATM phosphorylation was detected in CX-5461-incubated cells. Still, both gemcitabine and CX-5461 led to increased levels of CHK2 phosphorylation. Of note, CX-5461 has been previously reported to induce DNA damage, although the underlying mechanisms remain debated[17–20]. Overall, CX-5461 caused DNA damage, although its effect appeared distinct and less than that of gemcitabine after a 48-h incubation.

To confirm that the chemotherapeutic agents gemcitabine and CX-5461 exhibit anticancer effects, we assessed cell growth by performing crystal violet assays. As expected, both HeLa and MIA PaCa-2 were sensitive to gemcitabine and to increasing concentrations of CX-5461 (Figure 1(F,G)).

CX-5461 impacts on autophagy and lysosomal function

Next, we examined whether the nucleolar stressor CX-5461 could replicate the induction of autophagy and lysosomal function observed under gemcitabine treatment[2]. In HeLa cells, gemcitabine and CX-5461 exposure caused increased LC3B-II levels, along with elevated levels of phospho-ATG16L1 and mature CTSB, respective markers of newly formed autophagosomes[21] and lysosomal activity (Figure 2(A)). Additionally, accumulation of LC3B and LAMP1 puncta in gemcitabine- and CX-5461-incubated cells supported an increase in autophagosomes and expansion of the lysosomal network (Figure 2(B)). To accurately measure autophagy activity, we monitored autophagic flux by incubating cells with bafilomycin A1. LC3B-II and SQSTM1/p62 levels significantly increased in cells co-incubated with bafilomycin A1 and either gemcitabine or CX-5461 compared with the levels under bafilomycin A1 alone, indicating enhanced autophagy flux (Figure 2(C))[22]. Although CX-5461 significantly promoted autophagy flux in HeLa cells, the effect was inconsistent in MIA PaCa-2 cells (Figure 2(D)). One plausible explanation is that the elevated basal autophagy levels reported in pancreatic cancer cells mask subtle variations in autophagy flux, making these changes more difficult to detect and/or reach statistical significance[23]. Still, gemcitabine increased autophagic flux, aligning with our previous findings[2]. Notably, CX-5461 exhibited a similar efficacy to gemcitabine in increasing mature CTSB levels, indicative of increased lysosomal function (Figure 2(E)).

Figure 2.

Figure 2.

Open in a new tab

Autophagy and lysosomal function in response to CX-5461.

(A) HeLa cells were incubated for 48 h with DMSO (-), gemcitabine (10 μM), or CX-5461 (2 and 1 μM). Total cell lysates were analyzed by immunoblotting using anti-LC3B, p-ATG16L1S278, ATG16L1, CTSB, and GAPDH antibodies. Representative western blots are shown (left). (Right) Graphical representation of p-ATG16L1S278 (normalized to ATG16L1 or GAPDH) and mature CTSB (normalized to GAPDH) levels from biological replicates of three to four color-coded independent experiments (for DMSO, 10 μM gemcitabine, and 2 μM CX-5461). The ratio of protein of interest levels to loading control levels in control (DMSO) cells was set at 1. Data were analyzed using one-way ANOVA with Dunnett post-hoc tests. (B) HeLa cells were incubated for 24 h with DMSO (Control), gemcitabine (10 μM), or CX-5461 (2 μM) before autophagosome labeling using an anti-LC3B antibody or lysosome labeling using an anti-LAMP1 antibody. Nuclei were stained with DAPI. Scale bars, 10 μm. (C) HeLa cells were incubated for 48 h with vehicle (-), gemcitabine (10 μM), or CX-5461 (2 μM). Bafilomycin A1 (BafA1; 100 nM) or vehicle (DMSO) was added 4 h prior to cell lysis for autophagy flux measurement. Total cell lysates were immunoblotted as indicated (top). (Bottom) Graphical representation of LC3B-II levels (normalized to loading control; ACTIN or GAPDH) from six color-coded independent experiments. The ratio of LC3B-II to loading control levels in control DMSO cells was set at 1. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test. (D) MIA PaCa-2 cells were incubated for 48 h with vehicle (DMSO), gemcitabine (10 μM), or CX-5461 (2 and 1 μM). Bafilomycin A1 (BafA1; 100 nM) or vehicle (DMSO) was added 4 h prior to cell lysis for autophagy flux measurement (as in C). Total cell lysates were immunoblotted using anti-LC3B and ACTIN antibodies (top). (Bottom) Graphical representation of LC3B-II levels (normalized to ACTIN or GAPDH) from four color-coded independent experiments (for DMSO, 10 μM gemcitabine, and 2 μM CX-5461). The ratio of LC3B-II to loading control levels in control DMSO cells was set at 1. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test. (E) MIA PaCa-2 cells were incubated for 48 h with DMSO, gemcitabine (10 μM), or CX-5461 (2 and 1 μM). Total cell lysates were analyzed by immunoblotting using anti-p-ATG16L1S278, ATG16L1, CTSB, ACTIN and GAPDH antibodies (as in A). (Top) Representative western blots are shown. (Bottom) Graphical representation of p-ATG16L1S278 (normalized to ATG16L1 or ACTIN/GAPDH) and mature CTSB (normalized to GAPDH) levels from biological replicates of four color-coded independent experiments (for DMSO, 10 μM gemcitabine, and 2 μM CX-5461). The ratio of protein of interest levels to loading control levels in control (DMSO) cells was set at 1. Data were analyzed using one-way ANOVA with Dunnett post-hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001.

Our previous study demonstrated that gemcitabine promotes mature CTSB and lysosomal function through mechanisms dependent on TFEB[2]. Members of the microphthalmia/transcription factor E family, including TFEB and TFE3, respond to stress by undergoing changes in their phosphorylation status, enabling their accumulation in the nucleus to elicit a transcriptional program that promotes autophagy and lysosomal function[24]. To evaluate whether CX-5461 affects TFEB and TFE3, we analyzed their phosphorylation levels by examining their electrophoretic mobility on SDS-PAGE, a reliable method for detecting overall changes across the multiple phospho-sites of TFEB and TFE3[24]. Gemcitabine and CX-5461 led to an enrichment of the lower-molecular-weight forms of TFEB, indicative of changes in TFEB phosphorylation (Figure 3(A)). As previously reported for gemcitabine, the electrophoretic mobility pattern of TFEB in CX-5461-incubated cells was distinct from that of the cells exposed to Torin1, an mTOR inhibitor well known to mediate TFEB dephosphorylation[2,24]. Acceleration of the electrophoretic mobility of TFE3 was also detected in gemcitabine- and CX-5461-incubated cells, which was comparable to that induced by Torin1.

Figure 3.

Figure 3.

Open in a new tab

TFEB in response to CX-5461.

(A) HeLa and MIA PaCa-2 cells were incubated for 24 h with vehicle (DMSO, -), gemcitabine (G; 10 μM), CX-5461 (C; 2 μM) or Torin1 (T; 250 nM). Total cell lysates were analyzed by immunoblotting using the indicated antibodies. (B and C) HeLaTFEB-V5 cells were incubated for 24 h with DMSO (Control), gemcitabine (10 μM), or CX-5461 (1 μM) (B) or actinomycin D (5 nM) (C) before immunostaining using an anti-V5 antibody. Nuclei were stained with DAPI. Representative single Z-section images of TFEB-V5 are shown. Scale bars, 10 μm. HeLa (D) and MIA PaCa-2 (E) cells were incubated for 24 h with vehicle (-), gemcitabine (G; 10 μM), CX-5461 (C; 2 μM), or Torin1 (T; 250 nM). Representative immunoblots of equal amounts of cytoplasmic and nuclear-enriched fractions analyzed using anti-TFEB, GAPDH and HISTONE H3 antibodies. Graphical representation of nuclear TFEB levels (normalized to nuclear HISTONE H3) from three (MIA PaCa-2) to four (HeLa) color-coded independent experiments. The ratio of TFEB to HISTONE H3 levels in control DMSO cells was set at 1. Data were analyzed using unpaired t-tests. (F) Total cell lysates of HeLa and MIA PaCa-2 (MIA) cells stably depleted of TFEB (shTFEB) and control populations (shNT) were immunoblotted with the indicated antibodies. (G and H) (Top) Representative clonogenic assays for HeLashNT and HeLashTFEB (G), and MIAshNT and MIAshTFEB (H) cells pulse-treated 6 h with vehicle (DMSO, -), or the indicated concentration of gemcitabine or CX-5461. (Bottom) Scatter dot plot shows means ± SEM; N = 4–8 biological replicates from two to four color-coded independent experiments. Data were analyzed using two-way ANOVA with Tukey post-hoc tests. # when compared with vehicle-treated shNT cells, & when compared with vehicle-treated shTFEB, and * when compared as indicated. #, & p < 0.05, ##,&& p < 0.01, ####,&&&&,**** p < 0.0001.

To associate the alterations in electrophoretic mobility with the nuclear enrichment of TFEB, we conducted immunofluorescence and subcellular fractionation analyses. In HeLa cells stably expressing a tagged TFEB (TFEB-V5), TFEB staining was mainly localized to the nuclei upon exposure to gemcitabine or CX-5461, when compared to control cells depicting both cytosolic and nuclear staining (Figure 3(B)). The nucleolar stress inducer actinomycin D also promoted TFEB nuclear staining (Figure 3(C)). Additionally, subcellular fractionation revealed higher levels of the lower-molecular-weight forms of TFEB in the nuclear fraction of gemcitabine- or CX-5461-incubated HeLa and MIA PaCa-2 cells when compared to control cells (Figure 3(D,E)). Of note, we and others have reported high basal levels of nuclear TFEB in PDAC cells[2,25,26], which may account for the less pronounced fold change in nuclear TFEB observed in MIA PaCa-2 cells following gemcitabine and CX-5461 exposure (Figure 3(E)). Our results suggest that, similar to gemcitabine, CX-5461 promoted the nuclear abundance of TFEB.

Interference with TFEB sensitized cancer cells to CX-5461

To determine whether TFEB confers resistance to CX-5461 treatment, we used stable cell populations expressing an shRNA targeting TFEB. HeLashTFEB and MIAshTFEB cells displayed reduced TFEB levels (by approximately 50%) compared to their control counterparts (HeLashNT and MIAshNT cells) without pronounced changes in TFE3 levels (Figure 3(F)). HeLashTFEB and MIAshTFEB cells exhibited reduced clonogenic growth ability compared with that of HeLashNT and MIAshNT cells (by approximately 35% and 40% respectively) (Figure 3(G,H)), consistent with our published results for MIAshTFEB and PANC1shTFEB cells[2,25]. Gemcitabine and CX-5461 exposure further limited the clonogenic growth of HeLa and MIA cells. At the lowest CX-5461 concentration tested (5 and 10 nM for HeLa; 50 and 70 nM for MIA), shTFEB cells consistently displayed approximately 50% less clonogenic growth than their shNT counterparts, while at the highest CX-5461 concentration used (20 nM for HeLa and 100 nM for MIA), shTFEB cells had 60% less clonogenic growth ability than their shNT counterparts (Figure 3(G,H)). These results suggest that interference with TFEB sensitized cells to the chemotherapeutics gemcitabine and CX-5461, agents that induce nucleolar stress (Figure 1(A-C)).

Initial studies reported the induction of autophagy in response to CX-5461[10,27]. However, neither study directly measured autophagic flux, making the effect of CX-5461 on autophagy elusive. Additionally, in both studies, given that autophagy markers correlated with anti-proliferative effects or reduced viability, the autophagy response was associated with the cytotoxicity of CX-5461. In the present study, HeLa cells consistently exhibited increased autophagic flux in response to CX-5461, whereas the autophagy response was inconsistent in MIA PaCa-2 cells. Nonetheless, both cell lines showed cytotoxicity in response to CX-5461, as demonstrated by clonogenic growth and crystal violet assays, indicating that cytotoxicity may occur without a well-defined autophagy response. Therefore, our findings are more consistent with those of other studies suggesting that autophagy induction inhibits rather than enhances the anticancer effects of CX-5461[28,29].

Despite a variable autophagy response depending on the cancer cell lines used, the effect of CX-5461 on TFEB was highly reproducible. In fact, the acceleration of TFEB electromobility, suggestive of TFEB dephosphorylation, was consistent and observed at earlier time-points than the increase in autophagy flux. Although we did not test earlier time-points, TFEB dephosphorylation was reliably detected after 24 h of exposure to CX-5461 in all cell lines tested. Interfering with TFEB amplified the cytotoxic effects of CX-5461 and gemcitabine, suggesting that TFEB – perhaps beyond autophagy regulation – could serve as a broader readout of a protective response. Still, further experiments are needed to clarify the protective responses mediated by TFEB in cancer cells exposed to chemotherapeutic agents.

A recent study showed a cytoprotective role for TFEB against CX-3543, a small molecule disrupting NCL interaction with rDNA G-quadruplex complexes and consequently inhibiting RNA Pol I transcription[15,30]. Although CX-3543 acts distinctly from CX-5461 to inhibit RNA Pol I, the reported results emphasize a potential link between interference with RNA Pol I and TFEB regulation. Nevertheless, the mechanisms by which CX-5461 impacts TFEB remain to be determined. Although the primary target of CX-5461 (and CX-3543) is the initiation of rDNA transcription by RNA Pol I, some studies suggested that CX-5461 stabilizes G-quadruplexes and promotes topoisomerase poisoning[12,17,31]. Given that the concentration of CX-5461 used in our study is comparable to or lower than those reported to affect these targets, further work should clarify whether these reported effects of CX-5461 promote TFEB function.

In our study, both the TFEB-dependent protective response induced by the chemotherapeutic drugs gemcitabine and CX-5461 correlated with nucleolar stress. Since the RNA Pol I inhibitor CX-3543 also triggered nucleolar stress and a TFEB cytoprotective response, it suggests that TFEB may represent a protective signal against nucleolar stress[15]. Many protective signals induced by nucleolar stress have been attributed to p53, although p53-independent signals have also emerged[6,7]. In contrast to another study reporting p53-dependent protective signals in response to CX-5461[29], our study implies that this agent also induces resistance mechanisms without relying on functional p53. In this context, our findings suggest that another transcription factor, namely TFEB, may act as a sensor of nucleolar stress. Deciphering how nucleolar stress leads to TFEB activation will provide new interesting mechanistic insights into TFEB regulation. It may uncover previously unrecognized regulatory mechanisms, while determining whether established pathways like mTORC1, ERK or AMPK link nucleolar stress to TFEB regulation. The observed changes in TFEB electrophoretic mobility after gemcitabine and CX-4561 exposure, compared with Torin1, suggest that mTORC1-dependent mechanisms are unlikely to be involved, although further validation is needed.

Overall, in contrast to others suggesting that CX-5461 may bypass resistance mechanisms[12], our study showed that this promising anticancer agent induces a TFEB-dependent protective response. Since TFEB has been associated with chemoresistance mechanisms in response to gemcitabine and CX-3543[2,15], our findings emphasize the significance of exploring TFEB as an attractive strategy to improve the efficiency of various chemotherapeutic drugs, particularly those inducing nucleolar stress.

Materials and methods

Cell culture and reagents

A V5-tagged TFEB lentiviral expression vector was generated by Gateway LR recombination of pDONR223-TFEB (DNASU plasmid repository, HsCD00399586) into the destination vector pLX304 (gift from David Root; Addgene plasmid #25890) as previously done[32]. Lentiviruses were produced in HEK293T cells and used to infect HeLa cells to generate HeLaTFEB-V5 cells, as previously described[33]. A stable HeLaTFEB-V5 cell population was obtained after 10-days selection with 5 μg/mL Blasticidin S HCl (Wisent Bioproducts, 450–190-XL).

MIA PaCa-2 cells stably expressing a non-targeting shRNA (shNT) or shRNA targeting TFEB (shTFEB) were previously described[2]. HeLa cells stably expressing a non-targeting shRNA (shNT) or shRNA targeting TFEB (shTFEB) were generated as described for MIA PaCa-2[2,25].

HeLa and MIA PaCa-2 cells were cultured at 37°C under a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (Wisent Bioproducts, 319–005) supplemented with 10% fetal bovine serum (Wisent Bioproducts, 080–150), 10 mM HEPES (Wisent Bioproducts, 330–050), and 2 mM GlutaMAXTM (Thermo Fisher Scientific Inc., 35,050,061). For HeLa and MIA PaCa-2 shRNA stable populations, 2 μg/mL puromycin (Thermo Fisher Scientific Inc., A1113803) was added to the cell culture medium. Other reagents were purchased from: actinomycin D (Cell Signaling Technology, 15,021), gemcitabine (MedChemExpress, HY-B0003), CX-5461 (MedChemExpress, HY-13323A), Torin1 (Tocris, 4247), and bafilomycin A1 (Selleck Chemicals, S1413).

Crystal violet assays

HeLa (5 000 cells/well) and MIA PaCa-2 (10 000 cells/well) were seeded in 96-well plates. The following day, cells were incubated for 24 h with vehicle or the indicated concentrations of gemcitabine or CX-5461. The medium was then replaced, and cells were cultured for an additional 24 h. Subsequently, cells were rinsed with 1X PBS, stained for 20 min at room temperature with 0.5% crystal violet (Bio Basic, CB0331) in 20% methanol and washed with water. Plates were air-dried overnight, and the dye was solubilized in 10% acetic acid. Optic density (OD) was measured at 595 nm, with background OD from empty wells subtracted. OD values of untreated cells were set at 100%.

Immunoblotting

Cells were rinsed twice with ice-cold 1X PBS before being lysed in High Salt Buffer (1% Igepal, 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA pH 8.0, 10 mM NaF, 40 mM B-glycerophosphate, 10% glycerol, 0.5 µg/mL aprotinin, 0.5 µg/mL leupeptin and 0.5 µg/mL pepstatin, 1 mM PMSF, and 200 µM orthovanadate). Total cell lysates were sonicated and cleared by centrifugation (13,000 × g, 10 min, 4°C). The bicinchoninic acid (BCA) reagent procedure (Thermo Fisher Scientific Inc., 23,225) was used to measure protein concentrations, as recommended by the manufacturer.

For the enrichment of cytoplasmic and nuclear proteins, the Cell Fractionation Kit (New England Biolabs Ltd., 9038) was used as previously described[2], and as per the manufacturer’s instructions. Equal amounts of proteins were resolved on SDS-PAGE and transferred onto Amersham polyvinylidene fluoride (Sigma-Aldrich Canada Corp., 10,600,023) or nitrocellulose (Bio-Rad Laboratories Inc., 1,620,115) membranes. Membranes were probed, as previously described[2,25], using the following primary antibodies: ACTIN (Merck Millipore, MAB1501R), p-ATG16L1Ser278 (Cell Signaling Technology, 45,511), ATG16L1 (Cell Signaling Technology, 8089), p-ATMSer1981 (Cell Signaling Technology, 5883), ATM (Santa Cruz Biotechnology, 377,293), CATHEPSIN B (Cell Signaling Technology, 31,718), p-CHK2Thr68 (Cell Signaling Technology, 2197), CHK2 (Santa Cruz Biotechnology, 5278), p-DNA-PKcsSer2056 (Cell Signaling Technology, 68,716), DNA-PKcs (Santa Cruz Biotechnology, 390,849), GAPDH (Cell Signaling Technology, 2118), p-H2AXSer139 (H2AX) (Cell Signaling Technology, 9718), HISTONE H3 (Cell Signaling Technology, 3638), LC3B (Cell Signaling Technology, 3868), TFEB (Cell Signaling Technology, 4240), and TFE3 (Cell Signaling Technology, 81,744). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories Inc., 111–035-003 and 115–035-003, respectively).

Immunofluorescence and confocal microscopy

Cells were seeded on glass coverslips in a 6-well plate for 24 h before being treated as indicated. Treated cells were washed with cold PBS, fixed with cold 4% paraformaldehyde in PBS for 15 min, and permeabilized for 10 min with ice-cold (−20°C) 100% methanol or for 5 min in PBS containing 0.15% or 0.3% Triton X-100. Cells were rinsed twice with cold PBS then blocked for 45 min in PBS containing 5% FBS and 0.1% Tween before overnight incubation at 4°C with the primary antibody diluted in blocking solution. After three washes in PBS-0.1% Tween (PBS-T), the cells were incubated for 1 h at room temperature with the secondary antibodies. Cells were then washed in PBS-T, stained with DAPI, washed twice again, and mounted on slides using Fluoromount-G™ Mounting Medium (Invitrogen; 00495802). Images were acquired as described previously[2]. The following antibodies were used: LAMP1 (Santa Cruz Biotechnology, 20,011), LC3B (Cell Signaling Technology, 3868), NCL (Abcam, 136,649), NPM1 (Invitrogen FC-61991, 325,200), V5 Tag (Invitrogen SV5-Pk1, R960-25), and anti-rabbit or anti-mouse IgG (H+L) DyLight 488 or 549 (Vector Laboratories, DI1088 and DI2549).

Clonogenic assays

Stable populations of HeLa (250 cells/well) and MIA PaCa-2 (MIA; 500 cells/well) were seeded in 24-well plates overnight. Subsequently, the cells were incubated for 6 h with vehicle or the indicated concentrations of gemcitabine or CX-5461. After rinsing twice with culture media, the cells were cultured for 8–10 days. Thereafter, the cells were fixed for 10 min in 100% ice-cold methanol, stained with 0.5% crystal violet for 1 h, and washed with water. Colonies were scanned at a resolution of 600 pixels and counted using the ImageJ Colony_Area Plugin. The colony area was graphically represented, with the control cells set at 1.

Statistical analysis

Results were expressed as means ± standard error of the mean (SEM) unless otherwise stated in the figure legend. Data analysis was performed using GraphPad Prism 10.6.1. Differences between groups were assessed using Student’s t-test, one-way analysis of variance (ANOVA), or two-way ANOVA, as indicated in the figure legends.

Funding Statement

The work was supported by the Natural Sciences and Engineering Research Council of Canada [RGPIN-2016–05233]; Cancer Research Society and the Charlotte Légaré Memorial Fund [1281026].

Abbreviations

ANOVA

analysis of variance

NCL

nucleolin

NPM1

nucleophosmin

RNA Pol I

RNA polymerase I

TFEB

transcription factor EB

Data availability statement

Most of the data generated or analyzed in this study are included in this article. Data will be made available upon reasonable request to the corresponding author, provided the data will be used within the scope of the original informed consent.

Disclosure statement

The authors have no potential conflict of interest to declare.

References

  • 1.Khan SU, Fatima K, Aisha S, et al. Unveiling the mechanisms and challenges of cancer drug resistance. Cell Commun Signal. 2024;22(1):109. doi: 10.1186/s12964-023-01302-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Marchand B, Poulin MA, Lawson C.. Gemcitabine promotes autophagy and lysosomal function through ERK- and TFEB-dependent mechanisms. Cell Death Discov. 2023;9(1):45. doi: 10.1038/s41420-023-01342-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Poillet-Perez L, Sarry JE, Joffre C. Autophagy is a major metabolic regulator involved in cancer therapy resistance. Cell Rep. 2021;36(7):109528. doi: 10.1016/j.celrep.2021.109528 [DOI] [PubMed] [Google Scholar]
  • 4.Settembre C, Perera RM. Lysosomes as coordinators of cellular catabolism, metabolic signalling and organ physiology. Nat Rev Mol Cell Biol. 2023;25(3):223–10. doi: 10.1038/s41580-023-00676-x [DOI] [PubMed] [Google Scholar]
  • 5.de Sousa Cavalcante L, Monteiro G. Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur J Pharmacol. 2014;741:8–16. doi: 10.1016/j.ejphar.2014.07.041 [DOI] [PubMed] [Google Scholar]
  • 6.Boulon S, Westman BJ, Hutten S, et al. The nucleolus under stress. Mol Cell. 2010;40(2):216–227. doi: 10.1016/j.molcel.2010.09.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Gonzalez-Arzola K. The nucleolus: coordinating stress response and genomic stability. Biochim Biophys Acta Gene Regul Mech. 2024;1867(2):195029. doi: 10.1016/j.bbagrm.2024.195029 [DOI] [PubMed] [Google Scholar]
  • 8.Lafita-Navarro MC, Conacci-Sorrell M. Nucleolar stress: from development to cancer. Semin Cell Dev Biol. 2023;136:64–74. doi: 10.1016/j.semcdb.2022.04.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Potapova TA, Unruh JR, Conkright-Fincham J, et al. Distinct states of nucleolar stress induced by anticancer drugs. Elife. 2023:12. doi: 10.7554/eLife.88799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Drygin D, Lin A, Bliesath J, et al. Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth. Cancer Res. 2011;71(4):1418–1430. doi: 10.1158/0008-5472.CAN-10-1728 [DOI] [PubMed] [Google Scholar]
  • 11.Haddach M, Schwaebe MK, Michaux J, et al. Discovery of CX-5461, the first direct and selective inhibitor of RNA polymerase I, for cancer therapeutics. ACS Med Chem Lett. 2012;3(7):602–606. doi: 10.1021/ml300110s [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Li HX, He YM, Fei J, et al. The G-quadruplex ligand CX-5461: an innovative candidate for disease treatment. J Transl Med. 2025;23(1):457. doi: 10.1186/s12967-025-06473-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Corman A, Sirozh O, Lafarga V, et al. Targeting the nucleolus as a therapeutic strategy in human disease. Trends Biochem Sci. 2023;48(3):274–287. doi: 10.1016/j.tibs.2022.09.006 [DOI] [PubMed] [Google Scholar]
  • 14.Chen YT, Chen JJ, Wang HT. Targeting RNA polymerase I with hernandonine inhibits ribosomal RNA synthesis and tumor cell growth. Mol Cancer Res. 2019;17(11):2294–2305. doi: 10.1158/1541-7786.MCR-19-0402 [DOI] [PubMed] [Google Scholar]
  • 15.Ferret L, Pol JG, Sauvat A, et al. Lysosomal membrane permeabilization enhances the anticancer effects of POLR1 (RNA polymerase I) transcription inhibitors. Autophagy. 2025;21(10):2246–2265. doi: 10.1080/15548627.2025.2497614 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Groelly FJ, Fawkes M, Dagg RA, et al. Targeting DNA damage response pathways in cancer. Nat Rev Cancer. 2023;23(2):78–94. doi: 10.1038/s41568-022-00535-5 [DOI] [PubMed] [Google Scholar]
  • 17.Mars JC, Tremblay MG, Valere M, et al. The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability. NAR Cancer. 2020;2(4):zcaa032. doi: 10.1093/narcan/zcaa032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sanij E, Hannan KM, Xuan J, et al. CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer. Nat Commun. 2020;11(1):2641. doi: 10.1038/s41467-020-16393-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Snyers L, Laffer S, Lohnert R, et al. CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response. Sci Rep. 2022;12(1):13972. doi: 10.1038/s41598-022-17923-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Xu H, Di Antonio M, McKinney S, et al. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours. Nat Commun. 2017;8:14432. doi: 10.1038/ncomms14432 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Tian W, Alsaadi R, Guo Z, et al. An antibody for analysis of autophagy induction. Nat Methods. 2020;17(2):232–239. doi: 10.1038/s41592-019-0661-y [DOI] [PubMed] [Google Scholar]
  • 22.Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)(1). Autophagy. 2021;17(1):1–382. doi: 10.1080/15548627.2020.1797280 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yang S, Wang X, Contino G, et al. Pancreatic cancers require autophagy for tumor growth [Research support, N.I.H. extramural Research support, non-U.S. Gov’t]. Genes Dev. 2011;25(7):717–729. doi: 10.1101/gad.2016111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Puertollano R, Ferguson SM, Brugarolas J, et al. The complex relationship between TFEB transcription factor phosphorylation and subcellular localization. Embo J. 2018;37(11). doi: 10.15252/embj.201798804 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Marchand B, Arsenault D, Raymond-Fleury A. Glycogen synthase kinase-3 (GSK3) inhibition induces prosurvival autophagic signals in human pancreatic cancer cells. J Biol Chem. 2015;290(9):5592–5605. doi: 10.1074/jbc.M114.616714 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Perera RM, Stoykova S, Nicolay BN, et al. Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism. Nature. 2015;524(7565):361–365. doi: 10.1038/nature14587 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Li L, Li Y, Zhao J, et al. CX-5461 induces autophagy and inhibits tumor growth via mammalian target of rapamycin-related signaling pathways in osteosarcoma. Onco Targets Ther. 2016;9:5985–5997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Okamoto S, Miyano K, Kajikawa M, et al. The rRNA synthesis inhibitor CX-5461 may induce autophagy that inhibits anticancer drug-induced cell damage to leukemia cells. Biosci Biotechnol Biochem. 2020;84(11):2319–2326. doi: 10.1080/09168451.2020.1801378 [DOI] [PubMed] [Google Scholar]
  • 29.Liao H, Gaur A, Mauvais C, et al. p53 induces a survival transcriptional response after nucleolar stress. Mol Biol Cell. 2021;32(20):ar3. doi: 10.1091/mbc.E21-05-0251 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Drygin D, Siddiqui-Jain A, O’Brien S, et al. Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis. Cancer Res. 2009;69(19):7653–7661. doi: 10.1158/0008-5472.CAN-09-1304 [DOI] [PubMed] [Google Scholar]
  • 31.Bruno PM, Lu M, Dennis KA, et al. The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning. Proc Natl Acad Sci U S A. 2020;117(8):4053–4060. doi: 10.1073/pnas.1921649117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Blain J, Bedard J, Thompson M, et al. C-terminal deletion of NOTCH1 intracellular domain (N1ICD) increases its stability but does not amplify and recapitulate N1ICD-dependent signalling. Sci Rep. 2017;7(1):5034. doi: 10.1038/s41598-017-05119-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Marchand B, Tremblay I, Cagnol S. Inhibition of glycogen synthase kinase-3 activity triggers an apoptotic response in pancreatic cancer cells through JNK-dependent mechanisms [Research support, non-U.S. Gov’t]. Carcinogenesis. 2012;33(3):529–537. doi: 10.1093/carcin/bgr309 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Most of the data generated or analyzed in this study are included in this article. Data will be made available upon reasonable request to the corresponding author, provided the data will be used within the scope of the original informed consent.

Articles from Autophagy Reports are provided here courtesy of Taylor & Francis

RESOURCES