Researchers used RNA sequencing to analyze changes in gene expression after cabozantinib treatment in hepatocellular carcinoma cells. They also used a luciferase reporter assay to measure the transcriptional activity of transcription factor EB, or TFEB. Additional experiments examined the effects of cabozantinib alone or in combination with autophagy inhibitors, with findings assessed in vitro and in vivo. The study’s mechanistic and therapeutic experiments were conducted in preclinical models rather than in patients.

The mechanistic results indicated that cabozantinib increased the expression of acetyl-CoA synthetase 2, or ACSS2, and promoted its nuclear translocation. Nuclear ACSS2 was linked to activation of TFEB. TFEB then bound to promoter regions of lysosomal and autophagy-related genes, promoting lysosomal biogenesis and inducing autophagic flux. Thus, the experiments placed ACSS2 upstream of TFEB-dependent autophagy in the response of hepatocellular carcinoma cells to cabozantinib. The authors described this process as a protective response that counteracted the drug-induced inhibition of cell growth and apoptosis.

Impairing autophagy increased the cancer-cell killing associated with cabozantinib. Downregulating ACSS2 promoted cabozantinib-induced apoptosis, while treatment with an ACSS2 inhibitor enhanced the antitumor efficacy of cabozantinib in vitro and in vivo. Chloroquine produced a similar enhancement when used in combination with cabozantinib, supporting the conclusion that blocking autophagic flux can increase sensitivity to the treatment. These results were reported across both cell-based and animal experiments, although the supplied study abstract does not provide numerical response measures, statistical results, model details or other effect-size estimates.

The study proposes that cabozantinib can induce an ACSS2-TFEB-autophagy pathway that allows hepatocellular carcinoma cells to resist treatment, and that inhibiting ACSS2 or autophagy may counteract that response. The work offers a mechanistic basis for examining cabozantinib-based combinations in hepatocellular carcinoma. Its scope is preclinical: the reported evidence comes from cancer cells and animal models, not a clinical trial. Accordingly, the results show enhanced antitumor activity in experimental models and do not yet demonstrate that the combinations improve outcomes for patients with liver cancer.

Subject of Research: Preclinical enhancement of cabozantinib activity in hepatocellular carcinoma through ACSS2 and autophagy inhibition

Article Title: Inhibition of ACSS2 enhances cabozantinib sensitivity by blocking TFEB-induced autophagic flux in hepatocellular carcinoma

Article References: Feng, X., Lan, J., Du, J., Jiang, C., Qiu, G., Song, Y., Zhou, X., Zhang, Z., Xu, L., Hu, D., Liang, Y., Huang, J., Zeng, Y., & Yuan, K. (2026). Inhibition of ACSS2 enhances cabozantinib sensitivity by blocking TFEB-induced autophagic flux in hepatocellular carcinoma. British Journal of Cancer. https://doi.org/10.1038/s41416-026-03538-3

Image Credits: AI Generated

DOI: 10.1038/s41416-026-03538-3

Keywords: hepatocellular carcinoma, cabozantinib, ACSS2, TFEB, autophagic flux, drug resistance, chloroquine, lysosomal biogenesis, cancer cells, animal models, Inhibition, enhances

Autophagy is the cell’s housekeeping system, a process in which damaged proteins and organelles are engulfed in double-membraned vesicles called autophagosomes and delivered to lysosomes for destruction. Two molecular markers define this pathway: LC3, a protein that decorates the autophagosomal membrane, and p62, an adaptor that ferries ubiquitinated cargo to the growing autophagosome. The AAA-ATPase p97/VCP, meanwhile, is a hexameric molecular motor best known for extracting ubiquitinated proteins from cellular structures and handing them off to the proteasome or the autophagy machinery. Previous work by the same group had shown that blocking p97/VCP function in mouse Sertoli cells causes defective autophagosome maturation, cell cycle arrest, and apoptosis, making the protein a plausible candidate for regulating hormone-related protein turnover in these cells.

To test this idea, the researchers used the Sertoli 15P-1 cell line, a well-established mouse model derived through transmeiotic differentiation of male germ cells in culture. They exposed the cells to three pharmacological agents chosen for their opposing effects on autophagic flux. Testosterone served as the physiological ligand relevant to ABP regulation. Chloroquine, a lysosomotropic agent, blocks the fusion of autophagosomes with lysosomes, causing LC3 and p62 to accumulate because cargo can no longer be degraded. Rapamycin, by contrast, inhibits the mTOR signaling pathway and stimulates autophagy, accelerating the clearance of cellular components. Comparing the effects of these treatments allowed the team to infer whether ABP and p97/VCP travel together through the autophagic pipeline.

The experimental design combined three complementary techniques. Western blotting quantified protein expression levels across treatment groups, providing a biochemical readout of pathway activity. Single and double immunofluorescence staining revealed where p97/VCP, ABP, LC3, and p62 reside within the cells and, crucially, whether their locations overlap, a phenomenon called colocalization that suggests functional interaction. Finally, small interfering RNA (siRNA) transfection was used to specifically silence the gene encoding p97/VCP, creating a loss-of-function condition against which the drug treatments could be compared. This triangulation of methods strengthens the interpretation, because each approach has distinct limitations that the others can compensate for.

The Western blot results painted a picture consistent with autophagic involvement. p62 expression was highest in the group treated with both chloroquine and testosterone compared with the control siRNA condition, although the difference did not reach statistical significance. LC3 expression peaked in the chloroquine-treated group, again without reaching significance, which is the expected pattern when autophagic flux is blocked at the lysosomal step: autophagosomes accumulate because they cannot be cleared. As anticipated, the lowest expression of p97/VCP was detected in the VCPsi group, confirming that the siRNA transfection successfully knocked down the target protein and validating the loss-of-function arm of the study.

The immunofluorescence analyses delivered the study’s most striking findings. Significant differences in protein colocalization patterns emerged across all treatment groups, with one notable exception: p62 in rapamycin-treated samples showed no significant change. This pattern suggests that the physical association between p97/VCP and ABP shifts depending on the state of the autophagic machinery. When chloroquine jams the lysosomal bottleneck, proteins destined for degradation pile up and their colocalization with p97/VCP changes; when rapamycin revs up autophagy, the dynamics of interaction shift again. The authors report that these data provide the first evidence in the literature of a direct relationship between p97/VCP and ABP within Sertoli cells, a finding that reframes how scientists think about hormonal regulation in the testis.

Why does this matter for fertility? ABP’s job is to bind testosterone and maintain the extraordinarily high intratesticular concentrations that developing germ cells require. Disrupting ABP levels has measurable consequences: transgenic mice overexpressing ABP show meiotic arrest and germ cell apoptosis, and altered ABP expression has been linked to impaired sperm function in multiple models. If p97/VCP governs how quickly ABP is degraded, then the recycling motor effectively acts as a dial controlling how much ABP, and by extension how much bioavailable testosterone, is present in the seminiferous tubule environment at any given time. The data suggest a novel regulatory mechanism by which p97/VCP may influence spermatogenesis and male fertility through ABP degradation, offering a potential explanation for some forms of testicular dysfunction that currently lack a molecular diagnosis.

The implications extend well beyond the testis. ABP is not exclusive to Sertoli cells; it is also expressed in the brain, the liver, and the placenta, tissues where steroid hormone handling is equally critical. The authors propose that their work lays a foundational framework for future investigations in these ABP-expressing tissues, potentially linking p97/VCP expression to endocrine and pathophysiological disorders far removed from reproduction. Notably, p97/VCP has already been implicated in preeclamptic placenta, where altered expression coincides with impaired autophagy, and in testicular ischemia-reperfusion injury. Mutations in the VCP gene also cause multisystem proteinopathy, a devastating condition combining muscle, bone, and neurodegenerative disease, underscoring how central this protein is to cellular protein quality control across organ systems.

Technically, the study sits at the intersection of two major protein degradation systems. p97/VCP was originally identified as an ATPase co-purified with the inhibitor of NF-kappaB and the 26S proteasome, and it remains a linchpin of ubiquitin-proteasome-mediated degradation. But accumulating evidence shows that p97/VCP also cooperates with autophagy: in yeast, the orthologous Cdc48 protein regulates autophagosome biogenesis in concert with the ubiquitin-like protein Atg8, and in mammalian cells p97 inhibitors impair both ubiquitin-dependent and autophagic protein clearance. The p62 adaptor itself bridges the two systems, linking the autophagy pathway to the ubiquitin-proteasome system during the degradation of ubiquitinated proteins. The new findings place ABP squarely within this dual-disposal network in Sertoli cells.

As with any cell-line study, caveats apply. The statistical comparisons for p62 and LC3 did not reach significance, so the biochemical trends should be treated as suggestive rather than definitive, and the colocalization data, while significant, demonstrate proximity rather than direct biochemical binding. Follow-up work in primary Sertoli cells, intact seminiferous tubules, and animal models will be needed to confirm that the p97/VCP-ABP axis operates in vivo and to determine whether manipulating it can rescue fertility in disease settings. Nevertheless, by connecting a master regulator of protein homeostasis to a keystone hormone-binding protein, the study opens a concrete new avenue for research into male infertility, and it hints that therapies targeting autophagy, a strategy already being explored in fields from neurodegeneration to cancer, may one day find applications in reproductive medicine as well.

Subject of Research: Autophagic degradation of androgen-binding protein by p97/VCP in Sertoli cells and its implications for male fertility

Article Title: The Autophagic Interaction Between p97/VCP and ABP Protein Expressions in Sertoli Cell Line: the Effects of Chloroquine and Rapamycin

Article References: Corumluoglu, I., & Cayli, S. (2026). The Autophagic Interaction Between p97/VCP and ABP Protein Expressions in Sertoli Cell Line: the Effects of Chloroquine and Rapamycin. Reproductive Sciences. https://doi.org/10.1007/s43032-026-02231-3

Image Credits: AI Generated

DOI: 10.1007/s43032-026-02231-3

Keywords: p97/VCP, androgen-binding protein, autophagy, Sertoli cells, spermatogenesis, testosterone, chloroquine, rapamycin, male infertility, siRNA, LC3, p62

Drug-tolerant persister cells are a phenomenon increasingly recognized across many cancer types. These cells do not carry permanent resistance mutations; instead, they enter a reversible, slow-survival state that allows them to ride out treatment. Once chemotherapy stops, they can regrow into a full tumor, causing local recurrence that is often harder to treat than the original disease. In osteosarcoma, where aggressive multi-drug chemotherapy combined with surgery has improved survival significantly over past decades, relapse after apparently complete treatment remains a devastating clinical problem. The team behind the new study, led by researchers at the Eighth Affiliated Hospital of Sun Yat-sen University in Shenzhen, suspected that the unique biology of the bone microenvironment might be actively shaping this persister state rather than simply housing it.

The investigation began with a basic observation: when osteosarcoma cells were exposed to chemotherapy drugs in the laboratory, the surviving DTP cells showed consistently elevated levels of intracellular calcium. This finding immediately pointed toward the bone environment itself. Bone is, by definition, a mineral-rich tissue, saturated with calcium salts, and osteosarcoma cells live and evolve in that milieu. The researchers found that calcium buildup in persisting cells was accompanied by increased expression of ORAI3, a calcium channel protein involved in store-operated calcium entry, one of the main mechanisms by which cells replenish their internal calcium reserves. Elevated ORAI3 activity effectively opened the gates for calcium to flood into the persister cells, and that calcium flood turned out to be far from incidental.

Tracing the pathway downstream, the team showed that heightened calcium signaling promoted the nuclear translocation of NFAT4, a member of the nuclear factor of activated T cells family of transcription factors. Once inside the nucleus, NFAT4 acted on the gene encoding PINK1, PTEN-induced kinase 1, a well-known master regulator of mitophagy, the cellular process that identifies damaged mitochondria and directs their removal. In the persister cells, the result was a surge in PINK1-driven mitophagy. Rather than accumulating broken, dysfunctional mitochondria that would produce toxic reactive oxygen species and trigger cell death under chemotherapy stress, the DTP cells were continuously cleaning house, pruning away damaged mitochondria and preserving a pool of healthy ones.

The metabolic consequences of this mitochondrial quality control proved central to the survival strategy. Metabolic assays revealed that the persister cells maintained sustained tricarboxylic acid cycle activity and continued to rely on oxidative phosphorylation, the efficient mitochondrial process that generates cellular energy. This was a revealing finding, because many cancer cells under stress shift toward a less efficient, sugar-burning metabolism. The osteosarcoma persisters instead appeared to protect their oxidative powerhouses at all costs, and mitophagy was the mechanism that made this possible. By keeping mitochondrial homeostasis intact, the ORAI3–Ca²⁺–NFAT4–PINK1 axis ensured that the cells retained the energy production capacity needed to endure weeks of chemotherapeutic pressure without collapsing into metabolic failure.

To test whether this pathway mattered in living animals, the researchers engineered osteosarcoma xenografts carrying an inducible PINK1 knockdown system, allowing them to silence the gene at will. The results were striking in their timing. Before chemotherapy began, switching off PINK1 had no discernible effect on tumor growth; the cancer cells were perfectly viable. But once treatment started, tumors with silenced PINK1 showed reduced burden, and when chemotherapy was withdrawn, they regrew far more slowly than controls. In other words, PINK1 was not required for the tumor to exist, but it was essential for the tumor to survive chemotherapy and stage its comeback. Experiments in orthotopic bone tumors and lung metastasis models produced consistent results, reinforcing the conclusion that the pathway underpinned treatment tolerance across tumor settings.

The team then turned to pharmacological tools. GSK-7975 A, a small molecule that blocks ORAI-mediated calcium entry, proved capable of disrupting the axis at its source. Most notably, combining GSK-7975 A with chloroquine, an antimalarial drug that inhibits autophagic degradation, suppressed local recurrence in xenograft, orthotopic, and lung metastasis models. The logic of the combination is intuitive: the calcium channel blocker prevents the protective signal from being generated, while the autophagy inhibitor undermines the disposal machinery that persisters depend on. Together, the two agents deprived DTP cells of both the instruction and the equipment needed to maintain their mitochondrial shelter, leaving them vulnerable to chemotherapy they would otherwise survive.

Clinical evidence strengthened the case. Immunohistochemical analysis of human osteosarcoma tissues, comparing samples from patients who had received chemotherapy with those who had not, showed that ORAI3 and PINK1 expression was elevated after treatment, mirroring the laboratory findings. Single-cell and spatial transcriptomic analyses further supported the relevance of the axis in patient tumors, indicating that the calcium–mitophagy program is not merely a laboratory artifact but a feature of human disease. Given that both ORAI3 blockade and chloroquine-like compounds are already part of the pharmacological landscape, the pathway offers a plausible near-term target for clinical translation, potentially as an adjuvant strategy layered onto existing osteosarcoma chemotherapy regimens to eliminate the cells that seed recurrence.

The broader significance of the study lies in its reframing of the tumor microenvironment as an active participant in therapeutic failure. Rather than viewing recurrence simply as the outgrowth of resistant clones, the work suggests that bone’s defining biochemistry, its saturating calcium, hands osteosarcoma cells a ready-made survival program. It also connects two fields that rarely intersect: calcium channel biology and mitochondrial quality control, linked through NFAT4 as the molecular bridge. For a disease that disproportionately strikes adolescents and young adults, and where recurrence often carries a grim prognosis, the identification of a druggable axis that specifically disables persister cells during treatment represents a meaningful conceptual advance. If clinical studies confirm the animal findings, blocking ORAI3 or mitophagy could become a standard companion to chemotherapy, converting a treatment that currently leaves survivors vulnerable to relapse into one that clears the tumor down to its last resilient cell.

Subject of Research: A calcium-driven ORAI3–Ca²⁺–PINK1 mitophagy pathway that sustains drug-tolerant persister cells and local recurrence in osteosarcoma.

Article Title: Calcium-rich bone microenvironment promotes drug-tolerant persistence in osteosarcoma through ORAI3–Ca²⁺–PINK1–mitophagy axis

Article References: Li, H., Yu, H., Lu, Y., Liu, Y., Wu, W., Mi, R., Yang, W., Yuan, C., Yi, H., Gu, Y., Zhuang, J., & Ma, M. (2026). Calcium-rich bone microenvironment promotes drug-tolerant persistence in osteosarcoma through ORAI3–Ca²⁺–PINK1–mitophagy axis. Journal of Experimental & Clinical Cancer Research. https://doi.org/10.1186/s13046-026-03828-x

Image Credits: AI Generated

DOI: 10.1186/s13046-026-03828-x

Keywords: osteosarcoma, drug-tolerant persister cells, calcium signaling, ORAI3, PINK1, mitophagy, NFAT4, oxidative phosphorylation, local recurrence, chemotherapy, GSK-7975 A, chloroquine