HURP Silencing Differentially Impacts Spindle Architecture and Metastatic Behavior in Breast Cancer Cell Lines

Chromosomal instability (CIN) arising from mitotic errors is a hallmark of cancer progression, yet how specific spindle assembly factors are co-opted to support aggressive tumor phenotypes remains incompletely understood. Hepatoma Upregulated Protein (HURP/DLGAP5), a Ran-regulated microtubule-associated protein essential for kinetochore fiber stabilization and chromosome congression, is frequently overexpressed in aggressive cancers. Here, we investigated HURP’s role across a breast cancer metastatic gradient—immortalized MCF10A, the low-metastatic luminal T47D, and the highly metastatic triple-negative MDA-MB-231 cell lines—integrating quantitative spindle analysis, kinetochore tension measurements, spindle checkpoint profiling, migration dynamics, and three-dimensional spheroid modeling. We show that total HURP protein levels increase with metastatic potential, yet spindle-bound HURP is paradoxically reduced in MDA-MB-231 cells, indicating cytoplasmic mislocalization despite increased total protein levels. HURP silencing induced cell-line-specific defects: moderate disorganization and misorientation in MCF10A and T47D cells, but catastrophic spindle collapse, apoptosis, and G2/M arrest in MDA-MB-231 cells. Mechanistically, HURP depletion disrupted the spindle-associated levels and distributions of TPX2, Aurora-A, and NuMA in a subtype-dependent manner, implicating HURP as a context-dependent stabilizer of this mitotic regulatory axis. HURP loss reduced interkinetochore tension in all cell lines, but only MCF10A and T47D cells mounted a proportional BubR1-dependent checkpoint response; MDA-MB-231 cells showed reduced checkpoint signaling, consistent with constitutive spindle assembly checkpoint (SAC) attenuation in triple-negative breast cancer. Beyond mitosis, HURP depletion impaired collective migration and converted MDA-MB-231 cells from super-diffusive, amoeboid-like motility to sub-diffusive behavior, while minimally affecting the less aggressive cell lines. HURP-depleted MDA-MB-231 spheroids were significantly larger, less compact, and less spherical than controls, linking spindle regulation to tissue-level architectural coherence. These findings establish HURP as a multifunctional regulator coordinating mitotic fidelity, migration plasticity, and tumor architecture in breast cancer, with a selective dependency in highly metastatic cells, positioning it as a promising therapeutic target for aggressive breast cancers.