Ovarian cancer (OC) remains the deadliest gynecologic malignancy, with platinum-based chemotherapies such as carboplatin serving as the standard first-line treatment. However, the emergence of carboplatin resistance presents a major therapeutic challenge, and the underlying mechanisms remain incompletely understood. In this study, Dr. Tao Lu’s group from Indiana University School of Medicine, employed a novel validation-based insertional mutagenesis (VBIM) technique to identify genes that confer resistance to carboplatin in human epithelial OC cells. This screen revealed hematological and neurological expressed 1-like (HN1L/JPT2) as a previously unrecognized contributor to drug resistance. HN1L overexpression enhanced resistance to carboplatin, whereas shRNA-mediated knockdown sensitized OC cells to treatment. Mechanistically, HN1L promoted resistance by activating nuclear factor κB (NF-κB) signaling. In addition, HN1L depletion reduced anchorage-independent growth in vitro and impaired tumorigenicity in a xenograft model of OC. Immunohistochemical (IHC) analysis demonstrated elevated HN1L expression in both OC cell lines and patient tissues across multiple disease stages. These findings identify HN1L as a novel carboplatin resistance gene and suggest that targeting HN1L may represent a promising strategy for overcoming platinum resistance in OC.

The VBIM system utilized in this study leverages high-titer viral vectors for efficient genomic integration, leading to robust gene overexpression. OC cells infected with VBIM and exposed to carboplatin for two weeks yielded resistant colonies, indicating selection for mutation-driven survival. Surviving cells were isolated, and sequencing of VBIM-specific cDNA identified HN1L as the sole candidate gene. The resistant phenotype was reversed upon Cre recombinase treatment, confirming that the VBIM insertion into HN1L was causal. PCR analysis verified robust HN1L overexpression, validating its role in carboplatin resistance (Fig. 1).

To further assess HN1L’s function in OC chemoresistance, OC cells were genetically modified to either overexpress or knock down HN1L. Clonogenic assays demonstrated that HN1L-overexpressing cells were significantly more resistant to carboplatin, whereas HN1L knockdown cells displayed increased sensitivity. These results support a role for HN1L in enhancing carboplatin resistance in OC cells.

Given the established role of NF-κB signaling in chemoresistance, including resistance to platinum agents, the Lu Lab investigated whether HN1L modulates NF-κB activity. Luciferase reporter assays in OC and 293 cells revealed that HN1L overexpression significantly increased both basal and IL-1β–induced NF-κB activity. Conversely, HN1L knockdown suppressed NF-κB signaling. These findings indicate that HN1L is a potent activator of NF-κB.

Microarray analysis of 293 cells identified 818 NF-κB–responsive genes induced by IL-1β, of which 40% were further upregulated by HN1L. Notably, ~26% of these co-regulated genes have previously been implicated in platinum resistance. qPCR validation confirmed the upregulation of key NF-κB targets—including interleukin 1 receptor accessory protein (IL1RAP), major vault protein (MVP), and WNT1 inducible signaling pathway protein 1 (WISP1)—in HN1L-overexpressing cells treated with IL-1β. These genes are associated with inflammation, drug efflux, and tumor progression. Network analysis revealed that these targets are involved in DNA replication, repair, and metabolic pathways, with NF-κB positioned as a central regulatory hub.
To elucidate the mechanism of NF-κB activation, the Lu Lab examined IκBα degradation in HN1L-overexpressing cells. Western blotting showed that HN1L enhances NF-κB activity, potentially by promoting signaling events downstream of IκBα degradation.

Functionally, HN1L overexpression promoted anchorage-independent growth in soft agar, while knockdown suppressed it. In xenograft models, HN1L overexpression enhanced tumor growth and conferred resistance to carboplatin. IHC analysis of OC tissue microarrays (n = 100) and cell lines revealed significantly elevated HN1L expression across all clinical stages, reinforcing its role in OC progression and resistance.

Proteomic data from The Cancer Proteome Atlas (CPTAC) further confirmed significant HN1L overexpression in primary OC tissue compared to normal ovarian samples (P = 0.000029). Copy number analyses using GISTIC and cBioPortal revealed amplification of the HN1L gene in a substantial subset of OC cases. Additionally, UALCAN analysis showed increased HN1L expression across multiple cancer types, including colorectal and lung cancers. Gene amplification was a common alteration across these tumors.

To explore potential transcriptional regulation of HN1L, promoter analysis identified predicted binding motifs for E2F1, NFY, and EGR1. Notably, a putative NF-κB p50 binding site was located at -703 bp upstream of the transcription start site, raising the possibility of a feedforward regulatory loop in which NF-κB enhances HN1L transcription, further amplifying NF-κB signaling.

Together, these findings support a model (Fig. 2) wherein HN1L enhances carboplatin resistance by activating NF-κB signaling. Under normal conditions, IL-1β stimulation leads to IκBα degradation, permitting NF-κB nuclear translocation and activation of target genes. In the context of HN1L overexpression, this activation is amplified, resulting in upregulation of survival, DNA repair, and drug efflux genes that drive chemoresistance.

Importantly, these findings do not exclude the involvement of additional pathways. Whether HN1L modulates other oncogenic pathways such as PI3K/AKT or WNT signaling remains to be explored. For example, HN1—a homolog of HN1L—has been shown to regulate GSK3β phosphorylation via PI3K/AKT and is implicated in drug resistance. Notably, HN1 is also part of a four-gene panel used to distinguish OC from normal ovarian tissue, suggesting potential diagnostic utility.

The Lu Lab’s VBIM platform continues to serve as a powerful tool for uncovering genetic determinants of drug resistance. Previously, this approach identified NF-κB regulators such as FBXL11 and ARMC4. In the present study, VBIM revealed HN1L as a novel carboplatin resistance gene, expanding our understanding of resistance mechanisms in OC. HN1L has also been implicated in other cancers: in breast cancer, it promotes migration and correlates with poor prognosis, while its knockdown resensitizes triple-negative breast cancer to docetaxel. In non-small cell lung cancer (NSCLC), HN1L promotes cell cycle progression via the MAPK pathway, highlighting a conserved oncogenic function across tumor types.

The NF-κB target genes upregulated by HN1L play key roles in multiple cancer hallmarks. IL1RAP mediates inflammation and is overexpressed in both solid and hematologic malignancies. MVP is part of the vault complex and contributes to non–P-glycoprotein-mediated multidrug resistance. WISP1, a WNT pathway gene, has shown promise as a plasma biomarker for early OC detection in independent patient cohorts.

Interestingly, HN1L expression was highest in early-stage (I–II) OC and somewhat lower in late-stage (III–IV) disease. This suggests a role for HN1L in the early development of chemoresistance, though decreased expression at later stages may reflect influences from the tumor microenvironment or other regulatory factors. These observations warrant further investigation.

In summary, using the VBIM screening platform, the Lu Lab identified HN1L as a novel driver of carboplatin resistance in ovarian cancer. HN1L activates NF-κB signaling, promoting the expression of genes involved in survival and drug resistance. Acting downstream of IκBα, HN1L contributes to tumor progression both in vitro and in vivo. It is frequently overexpressed and amplified in OC and other cancers. Future studies should explore the potential NF-κB–HN1L feedback loop and examine whether HN1L modulates other oncogenic signaling pathways.

Collectively, these findings position HN1L as both a promising therapeutic target and a potential diagnostic biomarker. Targeting HN1L—alone or in combination with carboplatin—offers a compelling strategy to overcome platinum resistance in ovarian cancer and possibly other HN1L-driven malignancies.

Reference

Title of the original paper: Forward genetics identifies HN1L/JPT2 as a novel carboplatin resistance gene in ovarian cancer

Journal: Genes & Diseases
Genes & Diseases is a journal for molecular and translational medicine. The journal primarily focuses on publishing investigations on the molecular bases and experimental therapeutics of human diseases. Publication formats include full length research article, review article, short communication, correspondence, perspectives, commentary, views on news, and research watch.

DOI: https://doi.org/10.1016/j.gendis.2025.101720

Funding Information:

US National Institutes of Health (No. 1R01GM120156-01A1, R03CA223906-01, 1R03CA283225-A1),
Kay Yow Cancer Fund (V Foundation for Cancer Research) (USA) (No. 4486242),
Showalter Scholar fund from Indiana University School of Medicine (USA) (No. 2286263), Indiana University Melvin and Bren Simon Comprehensive Cancer Center (IUSCCC) fund (USA) (No. 2987664).

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