The monolayer (two-dimensional or 2D) cell culture, while widely used, lacks fidelity in replicating vital cell interactions seen in vivo, leading to a shift toward three-dimensional (3D) models. Although monolayers offer simplicity and cost-effectiveness, spheroids mimic cellular environments better. This is due to its nutrient gradients, which influence drug penetration and provide a more accurate reflection of clinical scenarios than monolayers. Consequently, 3D models are crucial in drug development, especially for anti-cancer therapeutics, enabling the screening of cell cycle inhibitors and combination therapies vital for heterogeneous tumor populations. Inhibiting processes like migration and invasion often require drugs targeting the cytoskeleton, which can exhibit dual functionality with cell cycle inhibitors. Therapeutic approaches with promising anti-cancer potential often exhibit reduced efficacy in 3D cell culture compared to their performance in monolayer settings, primarily due to the heightened complexity inherent in this system. In the face of this scenario, this review aims to survey existing knowledge on compounds utilized in both 2D and 3D cell cultures, assessing their responses across different culture types and discerning the implications for drug screening, particularly those impacting the cell cycle and cytoskeletal dynamics.

Precision oncology has revolutionized cancer biology, yet a critical disconnect persists: many pivotal oncogenic drivers, such as MYC, KRAS, and transcription factors, remain ‘undruggable’ by conventional small molecules due to a lack of traditional ligand-binding pockets. Overcoming this barrier is an urgent clinical necessity. This review maps and analyzes six key innovative strategies that are redefining druggability: (I) targeted protein degradation [TPD; proteolysis-targeting chimeras (PROTACs) and molecular glues], which catalytically eliminates proteins via the ubiquitin-proteasome system; (II) protein-protein interaction (PPI) inhibitors, which block critical oncogenic interfaces; (III) nucleic acid-based therapies [small interfering RNA (siRNA), antisense oligonucleotides (ASOs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)], which modulate targets at the RNA or DNA level; (IV) covalent inhibitors, which form irreversible bonds with specific residues; (V) allosteric modulation, which exploits hidden or cryptic binding pockets; and (VI) artificial intelligence (AI), which accelerates discovery by predicting structures and designing novel molecules. For each modality, we discuss mechanistic principles, historical development, and clinical translation, highlighting success stories and persistent challenges. Finally, we synthesize these approaches into an integrated perspective, arguing that their future convergence, for example, using AI to design next-generation degraders or combining PPI inhibitors with immunotherapies, will be essential to overcome the limitations of any single approach and reshape the standard of care. This review argues that the paradigm of undruggability is being dismantled by a shift from simple occupancy-based inhibition to event-driven degradation, state-specific modulation, and root-cause genetic correction.

SIVA1 is a multifunctional protein involved in processes that are critical for tumor progression. In breast cancer, SIVA1 is frequently overexpressed; however, its functional role remains poorly defined. This study aimed to elucidate the cellular mechanisms regulated by SIVA1 and to characterize its molecular network in breast cancer. Integrated in silico analyses and in vitro functional and molecular assays in breast cancer cell lines were conducted to investigate SIVA1 expression and associated signaling pathways. Cell proliferation, migration, invasion, and drug sensitivity were assessed in 2D and 3D culture models. Molecular analyses included markers of DNA damage and apoptosis, cytoskeletal regulators, ubiquitination status, and p53 signaling. The subcellular localization of SIVA1 was investigated. SIVA1 was overexpressed in breast cancer cells and associated with pathways related to protein secretion, spliceosome activity, DNA repair, and oxidative stress. Functional analyses linked SIVA1 to mitochondrial permeabilization, ubiquitin-mediated processes, cytoskeletal regulation, and p53-dependent apoptosis. SIVA1 knockdown increased cell growth, migration, and invasion, while enhancing sensitivity to paclitaxel in both 2D and 3D models, evidenced by reduced ATP levels and increased cleaved PARP1 and γ-H2AX. SIVA1 knockdown resulted in the accumulation of phosphorylated p53 and reduced phospho-Stathmin 1 and ubiquitin levels. SIVA1 displayed predominant nuclear localization, independent of treatment. In summary, these findings identify SIVA1 as a key regulator of proliferation and cytoskeletal dynamics in breast cancer, whose inhibition induces a more aggressive yet taxane-sensitive phenotype, with potential implications for patient stratification and therapeutic decision-making.