Scientists Reveal Potential Molecular Mechanism Driving Resistance to Therapy in Common Breast Cancer

Breast cancer is one of the most common malignancies in women worldwide, with approximately 70% of cases being estrogen receptor-positive (ER+) subtypes. In recent years, CDK4/6 inhibitors (such as palbociclib) combined with endocrine therapy (such as tamoxifen) have become first-line treatment options for ER+ breast cancer, significantly prolonging progression-free survival. However, with widespread clinical application, drug resistance has become increasingly prominent, with approximately 30%-50% of patients experiencing disease progression after 2-3 years of treatment. The mechanisms of drug resistance are complex and diverse, including Rb protein loss and Cyclin E1 amplification, but the causes of resistance in a large number of cases remain unclear.

Recently, in a study titled "JNK pathway suppression mediates insensitivity to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer," published in the Journal of Experimental & Clinical Cancer Research, scientists from the Sydney Garvan Institute of Medical Research and other institutions unexpectedly discovered, through genome-wide CRISPR/Cas9 screening, that inactivation of the JNK signaling pathway (specifically the MAP2K7 gene) may be a key factor in resistance to combination therapy. This discovery not only provides a new explanation for clinical drug resistance but also suggests that JNK pathway activity may serve as a biomarker for predicting efficacy.

Currently, research on CDK4/6 inhibitor resistance has largely focused on monotherapy models, while the mechanisms of resistance to combination endocrine therapy remain largely unknown. In this study, researchers systematically explored, for the first time, the genes driving resistance to the tamoxifen + palbociclib combination therapy in ER+ breast cancer cells using genome-wide CRISPR/Cas9 screening. The researchers hypothesized that the JNK pathway may influence drug sensitivity by regulating cellular stress responses and senescence. However, its role in ER+ breast cancer remains controversial. While some studies have shown that JNK activation promotes drug resistance, there is also evidence supporting its tumor suppressor role. To this end, the researchers designed a multifaceted experimental approach to validate the contradictory functions of the JNK pathway and its clinical relevance.

Using the ER+ breast cancer cell lines MCF-7 and T-47D as core models, CRISPR/Cas9 technology was used to generate cell lines with knockout genes for MAP2K7, MAPK8 (JNK1), and MAPK9 (JNK2). A genome-wide screen was performed using the Brunello library (targeting 19,114 genes), and sgRNA enrichment was analyzed by sequencing. In vivo experiments assessed metastatic burden using tail vein injection in immunodeficient mice. In vitro experiments included Western blotting (for detection of pJNK, c-JUN, etc.), RNA sequencing (for analysis of transcriptome changes), β-galactosidase staining (for assessment of senescence), and flow cytometry (for detection of apoptosis and cell cycle). Clinical data analysis integrated genomic and survival data from cohorts such as TCGA and METABRIC, and the prognostic value of pJNK protein expression was validated in a cohort of 78 patients with metastatic ER+ breast cancer.

Figure 1. CRISPR/Cas9 screens identify JNK pathway deficiency in endocrine therapy + CDK4/6 inhibitor insensitive ER+ breast cancer cells. (Alexandrou S, et al., 2025)

Key findings of this study:

1. CRISPR screening targeted the JNK pathway: MAP2K7 (an upstream kinase of JNK) deficiency was significantly enriched in drug-resistant cells, with a β score as high as 20.11, suggesting that JNK signaling inhibition is the primary cause of drug resistance.

2. Biological effects of MAP2K7 knockout: Knockout cells exhibited a 75%-97% reduction in pJNK phosphorylation and impaired stress-induced JNK activation. In a mouse model, MAP2K7 deficiency increased the number of lung metastases by 2-fold.

3. Mechanism of drug resistance: MAP2K7 deficiency downregulated expression of the AP-1 transcription factor c-JUN, impairing drug-induced senescence (a 50% reduction in β-galactosidase-positive cells), but did not affect apoptosis. RNA-seq revealed that JNK pathway-deficient cells exhibited a significantly reduced transcriptional response to combination therapy, particularly in E2F target genes and the G2/M checkpoint pathway.

4. Clinical Validation: TCGA data show that pJNK activity is reduced by 37.5% in patients with ER+ breast cancer who harbor MAP2K7 deficiency. In a metastatic cohort, patients with low pJNK expression had shorter progression-free survival. Furthermore, the Preoperative Palbociclib Treatment Trial (POP) demonstrated that patients with low JNK pathway expression had a poorer response to the drug's anti-proliferative effects.

This study reveals that the JNK pathway plays a dual role in ER+ breast cancer: when normally active, it exerts a tumor suppressive effect through c-JUN-mediated stress responses and senescence, while overactivation or complete inhibition leads to poor prognosis. MAP2K7 inactivation can evade the anti-proliferative effects of combination therapy by disrupting the AP-1/ERα transcriptional network, providing a novel target for clinical drug resistance. Future development of JNK pathway activity assays may optimize treatment options, and the exploration of MAP2K7 agonists is highly anticipated.

Reference

Alexandrou S, et al. JNK pathway suppression mediates insensitivity to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer[J]. Journal of Experimental & Clinical Cancer Research, 2025, 44(1): 244.