Tumour architecture inverts ARNT-dependent growth regulation and reveals CPE-mediated spheroid expansion in luminal breast cancer

Tumour architecture profoundly influences cancer cell behaviour, yet functional genomic studies predominantly rely on two-dimensional (2D) cultures. Here, we performed parallel chromosome 1q focused CRISPR screens in matched 2D monolayers and three-dimensional (3D) spheroid cultures of luminal breast cancer cells, which revealed ARNT as a prominent architecture-dependent fitness regulator. ARNT depletion reduced proliferation in 2D but enhanced spheroid growth in 3D. This functional switch was reproduced in MCF7 and T47D but not in nontumorigenic MCF12A cells, indicating a tumour and architecture dependent role of ARNT. Transcriptomic profiling revealed distinct responses to ARNT perturbation in 2D and 3D and pointed to carboxypeptidase E (CPE) as a downstream effector. ARNT modulation reciprocally altered CPE expression, and genetic epistasis established CPE as required for ARNT loss driven spheroid expansion and sufficient to restore growth in ARNT overexpressing spheroids. In primary tumours, ARNT and CPE were inversely correlated in low-hypoxia TCGA Luminal A samples, and single-cell RNA sequencing revealed an inverse association within ER positive cancer cells. Together, these findings demonstrate that the growth regulatory function of ARNT depends on tumour architecture and that a functional ARNT CPE axis controls luminal breast cancer spheroid growth, underscoring the importance of incorporating 3D context into functional genomic approaches.