Cancer mechanics is subjected to a wide variety of alterations during cancer progression among which the stiffness change, oftentimes tied to the level of cancer aggressivity, seems to be the most prominent. This study intends to uncover the mechanisms underlying such stiffness alterations between two prostate cancer cell lines 22Rv1 and PC-3 cells selected as model cell lines. To assess the differences in the cellular stiffness across the cell lines, the cells were subjected to several types of mechanical tests: Atomic Force Microscopy (AFM), Real-Time Deformability Cytometry (RT-DC), and shear flow-induced cell deformation. The protein profile uncovered significant differences in vimentin and actin content across the cell lines. Corroborating the experimental findings, we employed a structural Finite Element Model that accounts for the nucleus, cellular membrane, cytoplasm as well as cytoskeletal components: actin cortex and actin bundles to which the actin protein content was attributed, microtubules (tubulin content) and intermediate filaments (vimentin content); further details in Bansod, 2018. Reflecting the changes in the protein content in the computational setup under both local (AFM) and global (RT-DC and shear flow) mechanical loads uncovers, that the considered cytoskeletal structure is not capable of explaining fully the observed stiffness changes and another underlying mechanism is to be assumed playing a crucial role in the mechanical response. This points to questions about whether the different organization of filaments, different mechanical properties of the nucleus or potentially even some other organelles may play a role in cell stiffness.