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. 2026 Apr 24;11(17):25701–25711. doi: 10.1021/acsomega.6c00543

Proteotoxic Stress Triggers Autophagy-Mediated AS160 Degradation and Metabolic Reprogramming in Colorectal Cancer

Somrudee Reabroi †,‡,*, Nareerat Sutjarit §, Arthit Chairoungdua ‡,∥
PMCID: PMC13150765  PMID: 42110819

Abstract

AS160 (TBC1D4) is a key regulator of glucose transporter trafficking and is frequently overexpressed in several malignancies. However, the mechanisms regulating its protein stability and its contribution to colon tumor metabolism remain poorly understood. Here, we identify a proteotoxic stress-responsive mechanism regulating AS160 abundance that involves crosstalk between the ubiquitin-proteasome system (UPS), ER stress-related signaling, and autophagy. Paradoxically, proteasome inhibition with MG132 resulted in a dose-dependent reduction of AS160 protein levels in both HCT116 and HT29 colon cancer cells, accompanied by the induction of ER stress markers and autophagy activation, including p62 accumulation and increased LC3-II/LC3-I ratios. In contrast, blockade of lysosomal degradation with chloroquine (CHQ) led to marked AS160 accumulation, suggesting that autophagy drives AS160 turnover under proteotoxic stress. Alleviating ER stress with 4-phenylbutyric acid (4-PBA) or scavenging reactive oxygen species with N-acetylcysteine (NAC) did not prevent AS160 loss following MG132 treatment. Functionally, shRNA-mediated knockdown of AS160 did not significantly alter glucose uptake or total GLUT1 protein levels; however, it increased lactate secretion accompanied by reciprocal changes in lactate transporters including upregulation of the lactate transporter MCT4 and downregulation of MCT1. Overall, our results suggest that the stability of AS160 protein is regulated through autophagy-associated pathways and that reduced AS160 expression is associated with altered lactate handling. Targeting pathways that regulate AS160 stability may therefore represent a strategy to modulate tumor metabolic adaptation.

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1. Introduction

Akt substrate of 160 kDa (AS160), also known as TBC1D4, is a Rab-GTPase activating protein (RabGAP) that controls the intracellular trafficking of glucose transporter (GLUT)-containing vesicles. Originally identified as a downstream substrate of Akt, AS160 is required for basal retention of GLUT4 glucose transporter in adipocytes, , myotubes, − and cardiomyocytes. − Beyond its established role in glucose homeostasis, accumulating evidence suggests that AS160 is dysregulated in multiple human cancers and may contribute to cancer-associated cellular functions. Elevated expression and phosphorylation of AS160 have been reported in prostate, , breast, lung, and multiple myeloma, implicating AS160 in cancer progression. Functionally, AS160 has been documented to link to cancer cell proliferation and cell cycle progression, in part through the modulation of cell cycle regulators such as p21. Given the central role of glucose metabolism in supporting cancer cell growth and survival, AS160 has emerged as a potential mediator of metabolic regulation in cancers. However, despite growing interest in its cancer-associated roles, the biological functions of AS160 and the molecular mechanisms regulating its activity remain poorly understood. In particular, the upstream pathways controlling AS160 protein stability have not been thoroughly investigated.

Tumor cells frequently encounter proteotoxic stress due to high metabolic demand and nutrient deprivation, conditions that activate the endoplasmic reticulum (ER) stress response. ER stress arises from the accumulation of misfolded proteins within the ER lumen, triggering the unfolded protein response (UPR). , Under physiological conditions, ER stress sensors IRE1α, PERK, and ATF6 remain inactive through their association with the ER chaperone GRP78. Upon ER stress induction, GRP78 dissociates from these sensors to bind misfolded proteins, thereby activating UPR signaling pathways to restore proteostasis through attenuation of protein synthesis, enhancement of protein folding capacity, and activation of protein degradation mechanisms. , While the UPR is initially cytoprotective, supporting cancer cell survival in hostile microenvironments, excessive or prolonged ER stress can shift signaling toward growth inhibition or cell death. Consequently, the machinery governing protein quality control under ER stress represents a critical, yet complex, target for therapeutic intervention.

Protein quality control under ER stress is coordinated by multiple degradation systems, primarily the ubiquitin-proteasome system (UPS) and autophagy. The UPS mediates rapid clearance of misfolded and short-lived proteins, whereas autophagy facilitates the degradation of protein aggregates, damaged organelles, and long-lived proteins. − Increasing evidence indicates extensive crosstalk between these systems. Notably, when proteasomal capacity is compromised (proteotoxic stress), ER stress-induced autophagy often functions as a compensatory mechanism to maintain cellular homeostasis. , In cancer, autophagy exhibits context-dependent effects, either supporting tumor survival or suppressing tumor progression through the degradation of oncogenic proteins and the activation of apoptotic pathways. Genetic or functional disruption of key autophagy regulators, such as Beclin-1 and other autophagy-related (Atg) proteins, has been associated with increased tumorigenesis, whereas activation of autophagy has been shown to limit cancer cell growth through intrinsic apoptotic signaling or destabilization of oncogenic factors. − These suggest that proteotoxic stress-responsive degradation pathways are crucial determinants of protein stability in cancer cells, providing a mechanistic framework to investigate whether ER stress-autophagy-UPS crosstalk governs the regulation of AS160.

Based on these observations, we hypothesize that proteotoxic stress engages ER stress-associated autophagy as an upstream mechanism regulating AS160 protein abundance in colon cancer cells. To test this, we first define the signaling cascades connecting proteasome inhibition-induced ER stress to autophagic AS160 proteolysis. Second, we investigate the functional impact of AS160 on glucose metabolism in colorectal cancer. Because the molecular pathways controlling AS160 turnover in human malignancies remain unknown, this study addresses an important gap by linking proteotoxic stress-responsive degradation to metabolic reprogramming, providing new mechanistic insights into AS160 regulation in cancer cells.

2. Results

2.1. Proteasome Inhibition by MG132 Reduces AS160 Protein Levels and Activates ER Stress Responses

To illustrate the clinical relevance of AS160 expression in CRC, transcriptomic data from the GEPIA2 database were analyzed. As shown in Figure A, AS160 mRNA expression was significantly higher in colorectal adenocarcinoma tissues than in normal tissues, indicating that AS160 is upregulated in colorectal tumors. Experimental validation showed that MG132 reduced AS160 protein levels in a concentration-dependent manner in both HCT116 and HT29 cells, with a more pronounced and robust decrease observed in HCT116 (Figure B,C). Consistent with this, cell viability analysis indicated that HCT116 is more sensitive to MG132 than HT29 (Figure S1), which likely contributes to the stronger response at the same applied concentrations, while AS160 downregulation was also detectable at lower doses that did not cause marked cytotoxicity. Therefore, HCT116 cells were chosen for subsequent mechanistic experiments. This reduction of AS160 coincided with a marked accumulation of ubiquitinated proteins (Figure D), confirming effective proteasomal inhibition and induction of proteotoxic stress. In parallel, MG132 altered ER stress-associated signaling, including increased IRE1α, GRP78 and XBP1 splicing, in association with AS160 downregulation (Figure E). To distinguish whether AS160 loss was a direct consequence of ER stress, cells were treated with the canonical ER stress inducer tunicamycin (TM). Although TM robustly induced ER stress markers, it produced a weaker reduction in AS160 protein levels compared to MG132 at comparable concentrations (Figure F), suggesting that AS160 downregulation is more strongly linked to proteotoxic stress than to ER stress activation alone. Notably, TM produced weaker XBP 1s induction and AS160 reduction than MG132, consistent with its lower potency in reducing cell viability (Figure S2) and the distinct nature of ER specific unfolded protein accumulation compared with global proteotoxic stress imposed by proteasome inhibition. Collectively, these results indicate that AS160 is upregulated in colorectal tumors and that proteotoxic stress conditions engaging ER stress signaling are associated with reduced AS160 protein abundance.

1.

1

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Proteasome inhibition by MG132 reduces AS160 protein levels and activates ER stress responses. (A) Box plot analysis of AS160 (TBC1D4) mRNA expression in tumor and normal tissues from colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) cohorts derived from GEPIA2 (*p < 0.01 showed by the database default test). (B, C) Representative Western blot analysis showing AS160 protein levels in HCT116 (B) and HT29 (C) cells treated for 24 h with increasing concentrations of the proteasome inhibitor MG132, with tubulin used as a loading control. The corresponding densitometric quantification of AS160 protein levels normalized to tubulin is shown. (D) Representative Western blot analysis showing accumulation of ubiquitinated proteins following MG132 treatment, with tubulin used as a loading control. (E, F) Representative Western blot analysis showing ER stress-associated marker expression in HCT116 cells treated with increasing concentrations of the proteasome inhibitor MG132 (E) or TM (F), with tubulin used as a loading control. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined relative to the control group (*p < 0.05, **p < 0.01, ***p < 0.001).

2.2. Autophagy Inhibition Promotes AS160 Protein Accumulation

To clarify the mechanism underlying AS160 turnover, we compared the effects of proteasome inhibition with MG132 versus lysosomal inhibition with chloroquine (CHQ). Because MG132 reduced rather than preserved AS160 protein levels (Figure ), we reasoned that an alternative degradation route contributes to AS160 clearance. In a comparative dose–response analysis, MG132 produced a dose-dependent reduction of AS160 accompanied by elevated LC3-II expression (Figure A). While p62 levels increased at lower MG132 concentrations, the reduction observed at 5 μM likely reflects robust autophagic turnover of p62 as a substrate. These data are consistent with activation of autophagy-related responses under proteotoxic stress conditions. In contrast, CHQ induced a marked, dose-dependent accumulation of AS160, together with robust p62 and LC3-II accumulation consistent with effective blockade of lysosomal degradation and autophagic flux (Figure B). Bafilomycin A1 increased LC3-II and p62 but did not restore AS160 under MG132 treatment during an acute exposure period (Figure S9). To assess whether AS160 associates with autophagy machinery, FLAG-AS160 was immunoprecipitated and probed for p62. However, direct AS160-p62 interaction was not detected under basal conditions (Figure S4), suggesting that any association, if present, may be stress-dependent or transient. Taken together, these opposing outcomes support lysosomal involvement as a key determinant of AS160 abundance and indicate that AS160 stability is primarily controlled through the autophagy-associated and lysosomal pathway, particularly when proteostasis is impaired.

2.

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Autophagy inhibition promotes AS160 protein accumulation. Representative Western blot analysis of AS160, p62, and LC3 protein levels in HCT116 cells treated with increasing concentrations of MG132 (A) or CHQ (B) for 24 h. Tubulin was used as a loading control. The corresponding densitometric quantifications for AS160, p62, and the LC3-II/LC3-I ratio are shown. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined relative to the control group (*p < 0.05, ** < 0.01, ***p < 0.001).

2.3. Proteasome Inhibition Induces AS160 Downregulation in Association with ER Stress and Autophagy-Related Responses

To further define how proteotoxic stress influences AS160 stability, cells were treated with MG132 in the presence or absence of CHQ. In Figure A, MG132 reduced AS160 protein levels, whereas CHQ alone increased AS160 abundance. Co-treatment with CHQ partially restored AS160 relative to MG132 alone, supporting involvement of the autophagy pathway in AS160 loss under proteasome inhibition. These protein changes were accompanied by p62 accumulation and increased LC3-II to LC3-I ratios, consistent with altered autophagy associated responses. At the transcript level, MG132 significantly reduced AS160 mRNA expression, while CHQ increased AS160 mRNA expression and the combined treatment remained comparable to MG132 alone (Figure B). Consistent with stress responsive remodeling of the autophagy program, ATG5 and LC3B transcripts were significantly increased following individual MG132 or CHQ treatment, with the strongest induction observed under the combined treatment (Figure C). In parallel, MG132 increased ER stress marker transcripts including GRP78, IRE1α, and XBP 1s (Figure D). Confocal microscopy further showed altered spatial distribution of AS160 following MG132 exposure, with reduced signal intensity throughout the cell and increased ER-Tracker signal consistent with ER expansion during ER stress (Figure E). To provide context for these molecular analyses, cell viability was assessed under the same treatment conditions, showing a modest reduction with either MG132 or CHQ alone, and a more pronounced decrease upon cotreatment consistent with dual inhibition of proteasomal and lysosomal pathways (Figure S3).

3.

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Proteasome inhibition induces AS160 downregulation in association with ER stress and autophagy-related responses. (A) Representative Western blot analysis showing expression of AS160, p62, and LC3 in cells treated with vehicle (Control), 40 μM chloroquine (CHQ), 1 μM MG132, or the combination of 40 μM CHQ and 1 μM MG132, with tubulin used as a loading control. The corresponding densitometric analysis of AS160 and p62 protein levels normalized to tubulin, and the LC3-II/LC3-I ratio is shown. (B) Relative AS160 mRNA expression levels determined by RT-qPCR following the indicated treatments. (C) Relative mRNA expression levels of autophagy-related genes (ATG5 and LC3B) as determined by RT-qPCR. (D) Relative mRNA expression levels of ER stress markers (GRP78, IRE-1α, and XBP 1s) determined by RT-qPCR. (E) Representative confocal microscopy images showing the intracellular localization of AS160 (green) and the endoplasmic reticulum (stained with ER-Tracker Red). Merged images are shown to illustrate channel overlap. Magnification = 63×. (F) Representative Western blot analysis showing AS160 protein levels in cells treated with 1 μM MG132 in the presence or absence of 1 mM 4-PBA, with tubulin as a loading control. The corresponding densitometric quantification is shown. (G) Representative Western blot analysis showing AS160 protein levels in cells treated with 1 μM MG132 in the presence or absence of 5 mM NAC. The corresponding densitometric quantification is shown. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined relative to the control group (*p < 0.05, **p < 0.01, ***p < 0.001).

To determine whether AS160 degradation reflects a reversible consequence of ER stress or oxidative stress, cells were treated with MG132 and either the chemical chaperone 4-phenylbutyrate (4-PBA) or the ROS scavenger N-acetylcysteine (NAC). Neither 4-PBA nor NAC restored AS160 protein levels in MG132-treated cells (Figure F,G), indicating that AS160 downregulation under proteasome inhibition persists despite chemical chaperone or antioxidant cotreatment. Collectively, these results support a model in which proteotoxic stress promotes AS160 loss with autophagy involvement, while modulation of ER stress or ROS alone is insufficient to restore AS160 abundance under these conditions.

2.4. Effect of AS160 Knockdown on Glucose Metabolism

To investigate the functional role of AS160 in cancer cell metabolism, we established stable AS160-knockdown cells using two independent short hairpin RNA (shRNA) sequences. Western blot and RT-qPCR analysis confirmed a significant reduction in AS160 protein and mRNA levels (Figure A,B). We next examined the functional consequences of AS160 depletion on glucose metabolism. Despite the established role of AS160 in regulating glucose transport in insulin-sensitive tissues, AS160 knockdown in colon cancer cells did not significantly alter intracellular glucose uptake (Figure C). However, AS160 silencing resulted in a significant increase in lactate production (Figure D), indicating altered lactate handling under these conditions. To elucidate a potential mechanism driving this phenotype, we examined the expression of the lactate transporter MCT4. Consistent with the increased lactate secretion, MCT4 mRNA expression was significantly upregulated in AS160-depleted cells (Figure E). In addition, we observed reduced mRNA expression of the lactate importer MCT1, which may further contribute to the net lactate release phenotype (Figure F).

4.

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Effect of AS160 knockdown on glucose metabolism. (A) Representative Western blot analysis showing AS160 protein levels in HCT116 cells transduced with control shRNA (shControl) or two independent AS160-targeting shRNAs (shAS160–1 and shAS160–2), with tubulin used as a loading control. The corresponding densitometric analysis of AS160 protein levels normalized to tubulin is shown. (B) Relative AS160 mRNA expression levels determined by RT-qPCR following AS160 knockdown. (C) Glucose uptake measured in control and AS160 knockdown cells and expressed as a fold change relative to control. (D) Extracellular lactate production measured in conditioned media from control and AS160 knockdown cells and expressed as fold change relative to control. (E, F) Relative mRNA expression levels of MCT4 and MCT1 determined by RT-qPCR. (G) Representative Western blot analysis of GLUT1 protein levels in control and AS160 knockdown cells, with tubulin as a loading control. Multiple bands correspond to different glycosylated forms of GLUT1 protein. The corresponding densitometric quantification normalized to tubulin is shown. (H) Relative GLUT1 mRNA expression levels as determined by RT-qPCR. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined relative to shControl (*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant).

We further examined whether the preserved glucose uptake observed after AS160 knockdown was associated with compensatory regulation of glucose transporters. Given that GLUT4 expression was negligible in HCT116 cells compared to 3T3-L1 adipocyte controls (Figure S6), we focused on GLUT1, the predominant glucose transporter in certain types of cancer, including colon cancer. Although GLUT1 mRNA levels were reduced upon AS160 knockdown (Figure H), GLUT1 protein levels remained unchanged (Figure G), suggesting post-transcriptional regulation. To assess whether altered protein stability contributes to this observation, we performed cycloheximide (CHX) chase assays in shControl and AS160-knockdown cells. Over 48 h, GLUT1 protein turnover was comparable between groups with no significant differences observed, indicating that AS160 knockdown does not measurably affect GLUT1 protein stability (Figure S7). Collectively, these results indicate that AS160 depletion promotes a metabolic shift and lactate secretion characterized by increased lactate export via MCT4 and reduced import via MCT1 while preserving glucose uptake, potentially through stabilization of GLUT1 protein.

3. Discussion

AS160 is a well-established regulator of glucose transporter trafficking in insulin-sensitive tissues. ,,, Although accumulating evidence indicates that AS160 is dysregulated in several malignancies, ,, including colorectal cancer, the mechanisms governing its protein stability and its contribution to tumor metabolism remain unclear. Here, we demonstrate that AS160 protein abundance is sensitive to proteotoxic stress. Proteasome inhibition paradoxically promotes AS160 loss in association with ER stress induction and autophagy activation, while blockade of lysosomal degradation leads to AS160 accumulation. Functionally, AS160 depletion preserves glucose uptake but enhances lactate secretion by upregulating the lactate transporter MCT4 and downregulating the lactate importer MCT1, indicating a metabolic shift toward increased lactate export. These findings demonstrate that AS160 abundance is regulated by stress-responsive protein degradation pathways and reduced AS160 expression is functionally associated with altered lactate handling in cancer cells.

A canonical function of the UPS is the degradation of short-lived proteins, and inhibition of this pathway typically results in the accumulation of its specific substrates. , Accordingly, the reduction of AS160 protein levels following MG132 treatment is unexpected, as UPS blockade generally leads to the stabilization of proteasome substrates (Figure ). However, proteasome inhibition is known to induce proteotoxic stress through the accumulation of ubiquitinated proteins, which subsequently activate compensatory degradation pathways. We proposed that the observed loss of AS160 arises from a compensatory crosstalk between the proteasome and autophagy pathways, which is often triggered when the ubiquitin-proteasome system is overwhelmed or impaired. Under these conditions, cells frequently activate the autophagy-lysosome pathway as a secondary defense mechanism to eliminate ubiquitinated protein aggregates and maintain cellular homeostasis. In support of this interpretation, we observed that MG132 treatment increased LC3-II levels and altered p62 expression, indicating the activation of autophagy-associated responses during proteasome impairment (Figure A). Furthermore, the ability of the lysosomal inhibitor CHQ to partially restore AS160 levels in the presence of MG132 provides supportive evidence that the cell shunts AS160 to the lysosome for clearance when proteasomal function is compromised (Figure A,B). We also acknowledge that MG132 reduces AS160 mRNA levels (Figure B), indicating that transcriptional repression contributes to the overall protein loss. In addition, a CHX chase assay did not show a clear acceleration of AS160 loss in MG132-treated cells compared with control conditions, suggesting that AS160 turnover is relatively slow within the tested time window (Figure S8). Thus, proteotoxic stress reduces AS160 through both transcriptional downregulation and enhanced autophagy-associated degradation, with the post-translational component supported by the partial rescue observed with CHQ. Notably, alleviating ER stress with 4-PBA or scavenging ROS with NAC did not restore AS160 levels in MG132-treated cells (Figure F,G), indicating that AS160 loss under proteasome inhibition is not readily reversed by directly suppressing canonical ER stress signaling or oxidative stress. These data support a model in which AS160 is preferentially routed to compensatory autophagy-lysosome degradation when proteasomal function is compromised, highlighting functional crosstalk between the UPS and autophagy pathways in regulating AS160 stability.

Beyond its regulation by protein quality control pathways, we next investigated the functional consequences of AS160 on tumor glucose metabolism. Because AS160 is a RabGAP implicated in glucose transporter trafficking, ,, we asked whether AS160 depletion alters glucose transporter regulation in CRC cells. We focused on GLUT1 (SLC2A1) because it is the predominant glucose transporter in many tumors, and its overexpression is frequently associated with aggressive disease and a poor prognosis. , Importantly, we also assessed the expression of GLUT4 which is the canonical insulin-responsive transporter regulated by AS160 in muscle and adipose tissue. We found that GLUT4 expression was nearly undetectable in these colorectal cancer cells especially when compared to the high levels observed in mature 3T3-L1 adipocytes which confirms that GLUT1 rather than GLUT4 is the primary transporter mediating basal glucose entry in this specific cancer model (Figure S6). Metabolic reprogramming is a defining feature of cancer cells, yet the mechanisms that coordinate glucose utilization and lactate export remain incompletely understood. , Canonical studies in insulin-sensitive tissues have established AS160 as a primary regulator of GLUT4 translocation and glucose uptake, with loss of AS160 typically impairing glucose transport. ,, Our findings reveal a distinct metabolic role for AS160 in CRC cells, in which glucose uptake is uncoupled from lactate handling (Figure C,D). Specifically, shRNA-mediated depletion of AS160 did not significantly alter intracellular glucose uptake despite a marked reduction in GLUT1 mRNA. This preservation of glucose influx was accompanied by sustained GLUT1 protein levels, suggesting compensatory post-transcriptional mechanisms that maintain transporter availability in this context (Figure F). − In contrast, AS160 loss enhanced lactate secretion and upregulated the monocarboxylate transporter MCT4 while simultaneously downregulating MCT1 mRNA expression (Figure E,F). This reciprocal regulation of lactate transporters suggests that reduced AS160 expression is sufficient to increase lactate export while limiting its re-entry under basal culture conditions. Notably, increased lactate secretion does not necessarily imply a proportional increase in glucose-derived glycolysis as lactate can also arise from glutamine or other substrate-derived metabolism and then be exported. Given the established contribution of lactate efflux to microenvironmental acidification and pro-tumorigenic remodeling, − our data suggest that AS160 might restrain lactate export in CRC cells. These metabolic phenotypes were defined using stable shRNA knockdown to characterize AS160-dependent readouts independently of pharmacological stress, thereby minimizing confounding effects from proteasome- or lysosome inhibitor-associated cytotoxicity and off-target responses. Accordingly, the increased lactate secretion and MCT4 upregulation observed after AS160 depletion are interpreted as direct consequences of reduced AS160 expression.

However, we noted that the metabolic impact of pharmacological AS160 reduction via MG132 differed significantly from the shRNA-mediated knockdown results (Figure S5). Specifically, MG132 treatment led to a decrease in MCT4 mRNA and an increase in MCT1 mRNA which is the opposite of the trend observed in our stable knockdown model. This discrepancy suggests that the acute proteotoxic stress and broad transcriptional impact of proteasome inhibition may trigger distinct regulatory pathways that mask or override the metabolic changes caused by AS160 loss alone. For instance, MG132 is known to activate the Nrf2-mediated antioxidant response which has been reported to transcriptionally induce MCT1 expression to support metabolic flexibility and survival under conditions of high cellular stress. Consequently, the observed increase in MCT1 during MG132 exposure likely reflects a general stress-survival mechanism rather than a specific consequence of AS160 depletion. Similarly, the reduction in MCT4 mRNA following MG132 treatment may be attributed to the well-documented ability of proteasome inhibitors to cause nonspecific mRNA destabilization or global transcriptional repression of glycolytic gene programs during acute proteotoxic crises. These findings emphasize the importance of using stable genetic models to isolate the specific metabolic contributions of AS160 from the confounding off-target effects of pharmacological proteasome inhibitors. Whether proteotoxic stress-driven AS160 reduction elicits a comparable metabolic shift in other contexts remains an important question for future work and should be evaluated by measuring lactate handling under conditions that decrease AS160 via the stress-autophagy axis.

To contextualize these findings, analysis of the GEPIA2 data set indicates that AS160 is significantly overexpressed in colorectal tumors relative to normal tissues (Figure A). Although this upregulation appears discordant with our experimental observation that reduced AS160 expression is associated with increased lactate secretion, it may reflect the metabolic plasticity required during tumor progression. , We propose that this apparent paradox reflects context-dependent regulation of AS160 during distinct stages of tumor evolution. In early tumorigenesis and growth-permissive regions, increased glycolysis generates acidic byproducts and metabolic stress, and maintaining AS160 expression may provide a selective advantage by limiting excessive lactate efflux while preserving lactate import capacity. This aligns with our finding that AS160 depletion enhances lactate secretion and upregulates MCT4 while reducing MCT1, suggesting that elevated AS160 in tumors coordinates both lactate export and import to maintain metabolic balance under growth-permissive conditions (Figure ). From this perspective, elevated AS160 may help restrain excessive extracellular acidification while enabling retention or reutilization of lactate as a metabolic substrate or signaling molecule. However, as tumors progress and encounter more severe proteotoxic stresses including hypoxia and nutrient deprivation, compensatory pathways such as autophagy may be activated. Our MG132 might reflect this acute stress response, where broad proteotoxic insult triggers autophagy-associated degradation of AS160. The divergence between our genetic and pharmacological models supports this interpretation, as MG132-induced transcriptional changes reflect a global stress program distinct from the specific consequences of AS160 loss. Thus, while basal AS160 overexpression may benefit early tumors by coordinating lactate handling, acute proteotoxic stress encountered in advanced disease may override this regulation through autophagy-mediated degradation. Cancer cells must balance efficient energy production and lactate export with the retention of carbon substrates needed to support anabolic biosynthesis during rapid proliferation. , In this context, elevated basal AS160 expression in tumor cells may help limit excessive lactate efflux by maintaining lower MCT4 levels and higher MCT1 levels, thereby favoring glucose utilization for biomass accumulation under growth-permissive conditions. , Under proteotoxic stress, our data report that AS160 protein abundance becomes susceptible to autophagy-associated degradation, which might relieve this constraint and coincide with increased MCT4 expression and decreased MCT1 expression to drive net lactate export. Further studies in animal models will be needed to determine the physiological relevance of this AS160 regulation within tumors.

4. Conclusions

In conclusion, our study identifies AS160 as a stress-responsive protein whose abundance is regulated under conditions of proteostasis imbalance. Proteotoxic stress promotes the downregulation of AS160 in association with autophagy-lysosomal activity. In parallel, the functional depletion of AS160 does not significantly alter glucose uptake but increases lactate secretion together with the reciprocal regulation of lactate transporters characterized by the upregulation of MCT4 and the downregulation of MCT1. These findings suggest that AS160 regulation might be linked to metabolic changes by uncoupling glucose uptake from lactate handling. Overall, the data support a model in which proteostasis-linked regulation of AS160 influences metabolic adaptation in cancer cells by modulating the lactate export machinery. This study provides a basis for future studies to test whether targeting pathways that control AS160 stability can limit tumor metabolic plasticity.

5. Materials and Methods

5.1. Cell Culture and Reagents

Human colorectal cancer cell lines HCT116 (KRAS G13D mutant, p53 wild-type) and HT29 (BRAF V600E mutant, p53 R273H mutant) were obtained from the American Type Culture Collection (ATCC, USA). These lines were selected as established models for studying proteotoxic stress and metabolic remodeling in colorectal cancer as they represent distinct genetic backgrounds commonly found in clinical adenocarcinomas. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), and 1% penicillin-streptomycin (Gibco, USA). Cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2.

MG132, CHQ, and Bafilomycin A1 (Baf A1) were purchased from Selleck Chemicals (USA). 4-Phenylbutyrate (4-PBA), N-acetylcysteine (NAC), tunicamycin (TM), and cycloheximide (CHX) were obtained from Sigma-Aldrich (USA). All compounds were prepared as stock solutions according to the manufacturers’ instructions, stored as aliquots in −20 °C, and diluted in complete medium immediately before use. Unless otherwise indicated, cells were treated at the concentrations shown in the corresponding figure panels for 24 h. Time-dependent treatments are specified in the figure legends.

5.2. shRNA Constructs and Stable AS160 Knockdown

Lentiviral shRNA constructs targeting human AS160 and a control shRNA targeting EGFP (shControl), as well as the FLAG-tagged AS160 expression plasmid, were kindly gifted from Dr. Brian North’s laboratory (Creighton University, USA). The shRNA sense sequences were: #shAS160–1:5′-CTAACAACAAAGCCAAGATA-3′; and #shAS160–2:5′-GCTTTGATGAACAGATGACAA-3′.

5.3. Lentivirus Production and Transduction

Lentiviruses were produced in HEK293T cells by cotransfection of shRNA plasmids with psPAX2 and pMD2.G-VSV.G. Viral supernatants were collected 48 h post-transfection, centrifuged, and filtered through 0.45 μm membranes. Target cells were transduced in the presence of 10 μg/mL Polybrene, followed by puromycin selection for 48–72 h to generate stable populations. Knockdown efficiency was confirmed by Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting.

5.4. RNA Extraction and RT-qPCR

Total RNA was isolated using the NucleoSpin RNA extraction kit (Macherey-Nagel, Germany). Reverse transcription was performed using the iScript Select cDNA Synthesis Kit (Bio-Rad, USA). RT-qPCR was performed using SYBR Green Supermix (Bio-Rad) on a CFX Duet Real-Time PCR System (Bio-Rad, USA). Data were analyzed using CFX Maestro Software (Bio-Rad), and relative gene expression levels were calculated using the 2–ΔΔCt method normalized to the housekeeping gene GAPDH. Primer sequences are listed in Table S1.

5.5. Western Blotting

Cells were lysed in RIPA buffer (Cell Signaling Technology (CST), USA) supplemented with protease and phosphatase inhibitors. Lysates were resolved by 10–12% SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 5% nonfat milk. Membranes were probed overnight at 4 °C with primary antibodies against AS160, IRE1α, XBP1, GRP78, LC3B, p62, GLUT1, and tubulin (all from CST), followed by species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were developed using Luminata Crescendo Western HRP substrate (Merck, Germany) and imaged with a ChemiDoc Touch Imaging system (Bio-Rad). Band intensities were quantified using ImageJ software (National Institutes of Health, USA) and normalized to the corresponding tubulin loading control.

5.6. Immunoprecipitation

Cells were transfected with FLAG-tagged AS160 using the calcium phosphate method and harvested 48 h post-transfection. Lysates were prepared in lysis buffer (50 mM Tris–HCl, pH 7.5, 0.5 mM EDTA, 0.5% NP-40, and 150 mM NaCl) supplemented with protease and phosphatase inhibitors (CST), clarified by centrifugation, and incubated with anti-FLAG M2 agarose beads (Sigma) overnight at 4 °C with rotation. Beads were washed three times with lysis buffer, and eluted by boiling in sample buffer. Eluted proteins were analyzed by Western blotting as described above, and FLAG-tagged proteins were detected using an anti-FLAG antibody (Sigma).

5.7. Cycloheximide (CHX) Chase Assay

To assess protein stability, cells were treated with CHX at 50 μg/mL and harvested at the indicated time points. For AS160 stability under proteotoxic stress, cells were pretreated with MG132 at 1 μM before CHX treatment. For the genetic knockdown model, cells were treated with CHX and chased over time. Protein levels were analyzed by Western blotting as described above.

5.8. Immunofluorescence

Cells were seeded in 8-well chamber slides (SPL Life Sciences, Korea). For endoplasmic reticulum labeling, cells were fixed with 4% paraformaldehyde and stained using the ER-ID Red detection kit (Enzo Life Sciences, USA) according to the manufacturer’s protocol. For AS160 detection, samples were incubated overnight with anti-AS160 (CST), followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, USA). Nuclei were counterstained with Hoechst 33342. Images were acquired using a Zeiss LSM800 confocal microscope (Zeiss, Germany).

5.9. Glucose Uptake Assay

Intracellular glucose uptake was quantified using the Glucose Uptake Cell-Based Assay Kit (Cat no. 600470; Cayman Chemical, USA) according to the manufacturer’s protocol. Briefly, cells were cultured for 48 h, then glucose-deprived overnight and incubated with the fluorescent glucose analog 2-NBDG in glucose-free medium for 1 h. After cell washing, fluorescence intensity was measured immediately after the final wash at excitation and emission wavelengths of 488 and 535 nm, respectively, using a microplate reader.

5.10. Lactate Production Assay

Extracellular lactate levels were measured using the Lactate Assay Kit-WST (Dojindo Laboratories, Japan). Culture supernatants were harvested after 48 h of incubation and mixed with the WST working solution for analysis. Samples were incubated at 37 °C for 30 min, and absorbance was measured at 450 nm using a microplate reader.

5.11. Statistical Analysis

Gene expression analyses were performed using the Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) web server, which integrates RNA-sequencing data from TCGA and GTEx. Comparisons were performed for colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) cohorts using default parameters. Experimental data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s posthoc test (GraphPad Prism). P values are denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

Supplementary Material

ao6c00543_si_001.pdf (404.5KB, pdf)

Acknowledgments

This project is funded by National Research Council of Thailand (NRCT): Contract number N42A660979 awarded to S.R. The equipment for this research project is partially provided by the Faculty of Science, Mahidol University. The analytical instruments and facilities of this research project are in part supported by the Central Instrument Facility (CIF) Grant, Faculty of Science, Mahidol University.

Glossary

Abbreviations

AS160

Akt substrate of 160 kDa

UPS

ubiquitin-proteasome system

ER

endoplasmic reticulum

UPR

unfolded protein response

GRP78

glucose-regulated protein 78

IRE1α

inositol-requiring enzyme 1 alpha

XBP 1s

spliced X-box binding protein 1

LC3

microtubule-associated protein 1 light chain 3

CHQ

chloroquine

GLUT1

glucose transporter 1

MCT1

monocarboxylate transporter 1

MCT4

monocarboxylate transporter 4

CRC

colorectal cancer

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c00543.

  • qPCR primer sequences (Table S1); effect of MG132 on cell viability in HCT116 and HT-29 cells; effect of tunicamycin (TM) on cell viability in HCT116 cells; effects of MG132 and chloroquine (CHQ) on HCT116 cell viability; co-immunoprecipitation analysis of FLAG-AS160 with p62 under basal conditions; effects of MG132 and CHQ on MCT1 and MCT4 mRNA expression; GLUT4 mRNA expression in HCT116 cells and 3T3 L1 adipocytes; CHX chase analysis of GLUT1 proteinstability in control and AS160 knockdown cells; CHX chase analysis of AS160 protein stability under MG132 treatment; effect of bafilomycin A1 on MG132-induced AS160 downregulation (Figures S1–S9) (PDF)

S.R. Conceptualization, designed the research, performed the experiments, analyzed the data, and wrote the original draft of the manuscript; N.S. Performed the experiments and provided resources; A.C. Supervised project and provided acquisitions. All authors read and approved the final manuscript.

The authors declare no competing financial interest.

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