NDMA is one of the most extensively studied nitrosamine carcinogens. It appears in preserved and smoked foods, in contaminated drinking water, and it made international headlines when it was found in batches of the blood pressure drug valsartan and the heartburn medication ranitidine. The liver is its principal target organ, and prolonged low-dose exposure is known to predispose both animals and humans to hepatocellular carcinoma, the most common form of primary liver cancer, which accounts for roughly ninety percent of such cases and remains a leading cause of cancer mortality worldwide. Yet the question of whether NDMA does more than damage DNA — whether it can actively generate the self-renewing, treatment-resistant cells that make liver tumors so hard to eradicate — had remained unresolved.

To address this, the team, led by researchers at Nanjing Medical University, Ningbo No. 2 Hospital, and Sun Yat-sen University Cancer Center, exposed the non-cancerous human liver cell line LO2 to low concentrations of NDMA continuously for thirty passages, a period of about three months. The cells that received the higher dose, one microgram per milliliter, transformed dramatically. They became irregular and spindle-shaped, proliferated faster, migrated and invaded more aggressively than even the established liver cancer line Huh-7 used as a positive control, and showed elevated levels of hepatocellular carcinoma markers such as AFP, GGT1, and GSTP1 alongside a shift toward mesenchymal proteins like vimentin and N-cadherin, a hallmark of epithelial-mesenchymal transition. The researchers designated these cells TLO2 and used them as their experimental model of NDMA-driven liver cancer.

When the transformed cells were cultured in serum-free medium — a standard technique for enriching cancer stem cells — they formed large, abundant spherical clusters, a sign of self-renewal capacity. Flow cytometry revealed that roughly a quarter more cells expressed CD133, a well-established liver cancer stem cell marker, after this enrichment. Levels of additional stemness markers, including SOX2, OCT4, and EPCAM, rose in parallel. Soft agar and colony formation assays confirmed that the NDMA-transformed cells could grow without a solid substrate and form colonies at rates comparable to mature liver cancer cells. In short, chronic NDMA exposure did not merely convert liver cells into tumor-like cells; it endowed them with the stem-like properties that fuel metastasis, recurrence, and drug resistance.

The study then turned to ZNF652, a zinc finger transcription factor that previous work had linked to tumor suppression in several cancers. Database analyses using KM-PLOTTER and TNMplot showed that higher ZNF652 expression correlated with better survival in hepatocellular carcinoma patients, and that ZNF652 levels were lower in metastatic lesions than in primary tumors. In the NDMA-transformed cells, ZNF652 protein dropped after sphere enrichment. When the researchers forced ZNF652 back into the cells, spheres became smaller and less frequent, and the stemness markers CD133, SOX2, OCT4, and EPCAM all declined. Conversely, silencing ZNF652 with siRNA produced larger, more numerous spheres and higher marker expression. These results positioned ZNF652 as a brake on liver cancer stemness.

The Sonic Hedgehog pathway, a classical developmental signaling cascade that is frequently hijacked in cancer, emerged as the mechanism through which ZNF652 acts. Database comparisons showed that key components of the pathway — SHH, SMO, GLI1, and GLI2 — were elevated in liver cancer tissue relative to normal liver, and that they rose further when the transformed cells were enriched for stemness. GLI1, the transcriptional effector that translocates to the nucleus to activate Hedgehog target genes, proved particularly consequential: overexpressing it increased sphere size and frequency and boosted stemness markers, while knocking it down had the opposite effect. The pathway, in other words, actively promotes the cancer stem cell state in these cells.

The pivotal finding came when the team connected the two molecules. Correlation analyses revealed a negative relationship between ZNF652 and the Hedgehog components, and manipulating ZNF652 levels produced reciprocal changes in SHH, SMO, GLI1, and GLI2 expression. Using the JASPAR database, the researchers identified potential ZNF652 binding sites in the GLI1 promoter, then confirmed with dual-luciferase reporter assays that ZNF652 overexpression suppressed GLI1 transcriptional activity. Rescue experiments sealed the logic: when GLI1 was artificially raised, adding ZNF652 brought CD133 expression back down, and when ZNF652 was knocked down, reducing GLI1 reversed the resulting increase in stemness. ZNF652, the data showed, directly targets GLI1 to restrain the Hedgehog-driven cancer stem cell program.

Sulforaphane entered the picture as the pharmacological lever on this axis. The compound, produced when the enzyme myrosinase converts glucoraphanin in broccoli and other cruciferous vegetables, has long been known for anti-inflammatory, antioxidant, and anticancer properties, and ongoing clinical trials have explored its ability to inhibit cancer development. In the new study, treating the transformed cells with sulforaphane at concentrations of one, three, and five micromolar reduced sphere size and frequency, suppressed colony formation, and diminished self-renewal capacity as measured by extreme limiting dilution analysis. Western blots showed dose-dependent declines in CD133, SOX2, OCT4, and EPCAM, and immunofluorescence confirmed reduced CD133 signal. Crucially, sulforaphane also lowered the Hedgehog pathway components and raised ZNF652, and it reversed the stemness increase caused by either GLI1 overexpression or ZNF652 knockdown — evidence that the compound works through the same axis it was suspected of modulating.

To test whether these effects held in living tissue, the researchers implanted NDMA-pretreated Huh-7 cells subcutaneously into twenty female BALB/c nude mice, dividing them into control, NDMA-exposed, and two sulforaphane treatment groups receiving twelve point five or twenty-five milligrams per kilogram by oral gavage twice weekly for four weeks. Tumors in the NDMA group grew significantly larger than controls, while both sulforaphane doses reduced tumor volume and weight in a dose-dependent manner. Immunohistochemistry on excised tumors showed that NDMA exposure elevated CD133 and GLI1 while suppressing ZNF652, and that sulforaphane reversed all three changes, restoring ZNF652 and lowering the stemness and Hedgehog markers.

The authors acknowledge limitations: the xenograft model relied on cells pretreated with NDMA before implantation rather than systemic exposure in the animals, so it may not fully reproduce the natural course of NDMA-associated hepatocarcinogenesis or the complexity of the liver microenvironment, and the human database findings are correlative and were not validated in independent patient cohorts. Even so, the study offers a coherent mechanistic account of how a dietary carcinogen can generate the cellular seeds of liver cancer recurrence, and how a compound found in everyday vegetables can disrupt that process at its molecular root. If confirmed in clinical settings, targeting the ZNF652–GLI1 axis could open a new avenue for preventing or treating hepatocellular carcinoma, particularly in populations with high nitrosamine exposure.

Subject of Research: Sulforaphane inhibition of NDMA-induced hepatocellular carcinoma stem cell properties via the ZNF652/Sonic Hedgehog axis

Article Title: Sulforaphane inhibits N-nitrosodimethylamine induced hepatocellular carcinoma stem cell properties via ZNF652/Sonic hedgehog axis

Article References: Sulforaphane inhibits N-nitrosodimethylamine induced hepatocellular carcinoma stem cell properties via ZNF652/Sonic hedgehog axis. (n.d.). https://doi.org/10.3389/fcell.2026.1965529

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1965529

Keywords: sulforaphane, NDMA, hepatocellular carcinoma, cancer stem cells, ZNF652, Sonic Hedgehog, GLI1, broccoli, liver cancer, chemoprevention, CD133, nitrosamines