Functional genetic screens of chromosome 1q in breast cancer via 2D and 3D in vitro models

Breast cancer, with an incidence rate affecting approximately 2.3 million women globally, poses significant health challenges due to high recurrence rates and resistance to therapy. The diversity of molecular subtypes further complicates targeted treatment. Genetic abnormalities, particularly chromosomal alterations, play a critical role in tumour initiation and progression. Specifically, chromosome 1q (Chr1q) copy number alterations (CNA) are linked to poor patient prognosis and survival. This study hypotheses that Chr1q CNAs induce a genomic disorder/catastrophe, activating oncogenic drivers or causing gene expression loss. To investigate, we performed functional CRISPR screening targeting Chr1q genes in breast cancer. We utilised both 2D and 3D cultures in two breast cancer cell lines and one normal breast cell line to capture cancer-specific phenotypes influencing proliferation. We identified cancer-specific dropout genes negatively impacting proliferation in breast cancer cells but not in non-tumorigenic controls. Among 14 shared candidates, RPRD2 and GON4L were selected for further validation due to their significant effects on breast cancer proliferation in both 2D and 3D models. RPRD2 showed cancer-specific activity, while GON4L also impacted normal breast cells. For enriched candidates with positive phenotypes, we normalised 3D screening results by 2D (3D/2D). ARL8A and ARNT emerged as significant in at least one breast cancer cell line. Validation experiments revealed that blocking ARL8A and ARNT decreased proliferation in 2D models but increased 3D spheroid growth, indicating differential phenotypic impacts. Additionally, ARNT knockout in normal breast cells led to increased proliferation. This study highlights the necessity of transitioning from 2D to 3D models in functional screening for more accurate phenotypic evaluations. It also underscores the importance of Chr1q in breast cancer carcinogenesis and identifies potential cancer driver genes.