Persistent ATF6α Activation Drives Liver Cancer Through Metabolic Reprogramming

Hepatocellular carcinoma (HCC) is a leading cause of cancer death with limited treatment options. The unfolded protein response (UPR), a cellular reaction to stress in the endoplasmic reticulum (ER), is known to be involved in HCC. However, the specific role of the UPR protein ATF6α has been unclear. This research reveals that chronic activation of ATF6α acts as a driver of liver tumours and a master regulator of metabolism, suppressing the body's immune surveillance. This is in stark contrast to its typical role as a protective response to short-term stress.

In human patients, high ATF6α activity correlates with aggressive cancer, shorter survival, and a suppressed local immune environment. Experiments in mice confirmed that activating ATF6α specifically in liver cells caused progressive liver inflammation with ER stress, immunosuppression, and cell overgrowth. This activation increased sugar breakdown (glycolysis) while directly repressing a key sugar-making enzyme, FBP1. Restoring FBP1 expression reversed many of the harmful effects. Long-term ATF6α activation triggered liver cancer, characterized by exhausted immune T cells within the tumour. Blocking immune checkpoints restored immune function and reduced cancer growth. Patients who responded completely to immunotherapy had significantly higher ATF6α activity than those with weaker responses.

Furthermore, genetically removing ATF6α or treating with targeted genetic therapies reduced liver cancer in preclinical models. Therefore, prolonged ATF6α activation drives ER stress, leading to an immunosuppressive state fueled by altered sugar metabolism, which also makes tumours more sensitive to immune checkpoint therapy. These findings suggest that persistently active ATF6α is a tumour driver, a potential marker to predict immunotherapy response, and a promising new drug target for HCC.

HCC makes up most primary liver cancers and usually develops from damaged liver cells in chronic liver disease. While new immunotherapies have improved survival, the complex interactions between genetics, metabolism, and inflammation remain a major hurdle. T cells that enter liver tumours often become exhausted, leading to poor outcomes. A standard treatment for advanced HCC uses a combination of an immune checkpoint blocker (atezolizumab) and a drug that blocks blood vessel growth (bevacizumab). Metabolic changes in the tumour, including glucose deprivation, are thought to weaken anti-cancer therapies and promote tumour growth, highlighting the need for new strategies.

ER stress and UPR activation are negative indicators in many cancers, including liver disease. The UPR has three main branches: PERK, IRE1α, and ATF6α. When activated, ATF6α is cut to release a fragment (nATF6α) that moves to the cell nucleus to turn on genes for ER repair. While PERK and IRE1α can promote cancer, less is known about ATF6α. This study shows that, unlike its protective role in acute stress, chronic ATF6α activation becomes a cancer driver that controls glucose metabolism and contributes to immunosuppression.

Analysis of human liver cancer samples showed that ATF6α activity was consistently higher in tumours compared to healthy liver tissue. In large patient databases, a signature of high ATF6α activity was strongly linked to reduced patient survival. This connection appeared unique to liver cancer, as the same signature did not predict survival in other major cancer types.

Detailed examination of tissue samples revealed that tumours with high ATF6α activity were often higher grade (more aggressive). Protein analysis confirmed that the active form of ATF6α accumulated specifically in the tumours. In patients, the active protein was found in the nuclei of liver cells at the edge of tumours.

Advanced spatial analysis of tumour regions with high versus low ATF6α revealed distinct genetic profiles. High ATF6α areas showed increased activity in pathways related to immune checkpoints (PD-1/PD-L1, CTLA-4), low oxygen (hypoxia), cell division, and glycolysis. This high glycolytic activity was linked to low levels of the FBP1 enzyme. Low FBP1 is a known marker of an aggressive HCC subtype and poorer survival.

Further cellular imaging showed that tumours with high ATF6α had more infiltrating immune cells, particularly CD8 T cells. However, these T cells showed high levels of exhaustion markers like PD-1 and TIM3. Analysis of immune cell neighborhoods showed these exhausted T cells clustered with other suppressive immune cells, creating a local immunosuppressive environment. Notably, a subtype of T cells (TCF1+) known to respond well to immunotherapy did not cluster in these suppressive areas in high ATF6α tumours.

This suggested a mechanism for why high ATF6α tumours might be more sensitive to immunotherapy. Supporting this, a study of patients with advanced HCC treated with anti-PD-1 therapy found that those who had a complete response had significantly higher expression of ATF6α target genes.

To test ATF6α's role, researchers created a mouse model with hepatocyte-specific activation of ATF6α. These mice developed enlarged livers and liver damage, evident by elevated blood markers (ALT, AST). Electron microscopy confirmed the livers were under ER stress, showing swollen and disrupted ER structures. Molecular analysis confirmed activation of the ATF6α pathway and its targets.

Activation led to increased liver cell proliferation, DNA damage, and cell death markers, alongside signs of early cancer development. Genetic profiling showed upregulation of pathways for the UPR, glycolysis, inflammation, and cell division, while pathways for detoxification and energy production (oxidative phosphorylation) were down.

Crucially, ATF6α activation increased enzymes for glycolysis but suppressed key enzymes for making new glucose (gluconeogenesis), including FBP1. This led to depleted liver glycogen and glucose stores.

A key experiment using chromatin analysis (CUT&RUN and ATAC-seq) demonstrated that the active nATF6α protein directly binds to the regulatory region of the Fbp1 gene. This binding was associated with reduced chromatin accessibility at the Fbp1 promoter, effectively shutting down its expression.

Mice with persistent ATF6α activation had altered glucose metabolism. To test if loss of FBP1 was central to the damage, researchers used a virus to restore FBP1 expression in the livers of these mice.

Restoring FBP1 significantly reduced liver enlargement, injury markers, and glucose intolerance. It also lessened ER stress, DNA damage, and cell overgrowth caused by ATF6α activation. Genetically, it shifted metabolism away from glycolysis back towards oxidative phosphorylation, increased glucose levels, and reduced the uptake of a labeled fuel source (lactate).

Experiments with different versions of the FBP1 enzyme showed that its catalytic activity—its ability to perform its chemical function in gluconeogenesis—was necessary to restore glycogen and mitigate the effects of ATF6α activation. The data suggest a vicious cycle: ATF6α activation represses FBP1, which blocks gluconeogenesis and accelerates glycolysis. This depletes glucose/glycogen needed for proper protein processing in the ER, which in turn worsens ER stress and further activates ATF6α.

Given that chronic liver injury progresses to cancer, the long-term effects were studied. Mice with hepatocyte-specific ATF6α activation had reduced lifespan and body weight due to the spontaneous development of liver tumours. By 12 months of age, most of these mice had developed HCC, while control mice did not.

These mouse tumours mirrored human disease, showing markers of cancer stem cells, DNA damage, and activation of cancer-promoting pathways. Analysis of the tumour immune environment revealed a massive infiltration of T cells, but these cells were in a profoundly exhausted state, expressing multiple inhibitory receptors. This state of nutrient deprivation and exhaustion created a highly immunosuppressive tumour.

Because the tumours had many exhausted T cells, researchers tested immune checkpoint blockade (ICB). Treating tumour-bearing mice with antibodies against PD-1 and CTLA-4 led to a striking reduction in tumour burden and significantly improved survival. The therapy reinvigorated the exhausted T cells, increasing their numbers and cytotoxic function within the tumours.

Mechanistically, the effectiveness of ICB was linked to the metabolic state. The ATF6α-driven, glycolysis-heavy environment depleted key nutrients like glucose, which directly contributed to T cell exhaustion. Restoring FBP1 expression, which normalizes glucose metabolism, also enhanced the anti-tumour immune response and improved the efficacy of immunotherapy.

Finally, the study explored ATF6α as a direct therapeutic target. In several established mouse models of liver cancer (induced by chemicals or high-fat diet), reducing ATF6α activity was beneficial. This was achieved in three ways: 1) complete genetic knockout, 2) deleting ATF6α specifically in liver cells after tumours formed, and 3) using a therapeutic injection of antisense oligonucleotides (ASOs) designed to degrade Atf6 mRNA in the liver.

All three approaches significantly reduced liver tumour growth. The ASO treatment, a clinically relevant method, was particularly effective, reducing tumour progression and markers of cancer aggressiveness. This demonstrates the potential of targeting the ATF6α pathway itself for liver cancer treatment.

This work redefines ATF6α from a short-term adaptive protein to a central driver of liver cancer when chronically activated. It establishes a direct mechanistic chain: persistent ATF6α activation → direct repression of FBP1 → a metabolic shift to glycolysis → depletion of glucose/glycogen → worsened ER stress and promotion of tumour growth. This metabolic reprogramming also creates an immunosuppressive tumour environment by exhausting infiltrating T cells.

Paradoxically, this exhausted state makes the tumours more vulnerable to immune checkpoint inhibitors, identifying high ATF6α activity as a potential biomarker for predicting which HCC patients will best respond to immunotherapy. Furthermore, directly targeting the ATF6α pathway itself, for example with hepatocyte-directed ASOs, emerges as a promising new therapeutic strategy, potentially to be used alone or in combination with existing immunotherapies.