Silencing of Profilin-1 suppresses cell adhesion and tumor growth via predicted alterations in integrin and Ca2+ signaling in T24M-based bladder cancer models

Abstract

Bladder cancer (BC) is the second most common malignancy of the genitourinary system, characterized by the highest recurrence rate of all cancers. Treatment options are limited; thus a thorough understanding of the underlying molecular mechanisms is needed to guide the discovery of novel therapeutic targets. Profilins are actin binding proteins with attributed pleiotropic functions to cytoskeletal remodeling, cell adhesion, motility, even transcriptional regulation, not fully characterized yet. Earlier studies from our laboratory revealed that decreased tissue levels of Profilin-1 (PFN1) are correlated with BC progression to muscle invasive disease. Herein, we describe a comprehensive analysis of PFN1 silencing via shRNA, in vitro (by employing T24M cells) and in vivo [(with T24M xenografts in non-obese diabetic severe combined immunodeficient mice (NOD/SCID) mice]. A combination of phenotypic and molecular assays, including migration, proliferation, adhesion assays, flow cytometry and total mRNA sequencing, as well as immunohistochemistry for investigation of selected findings in human specimens were applied. A decrease in BC cell adhesion and tumor growth in vivo following PFN downregulation are observed, likely associated with the concomitant downregulation of Fibronectin receptor, Endothelin-1, and Actin polymerization. A decrease in the levels of multiple key members of the non-canonical Wnt/Ca2+ signaling pathway is also detected following PFN1 suppression, providing the groundwork for future studies, addressing the specific role of PFN1 in Ca2+ signaling, particularly in the muscle invasive disease.

Profilin 1–mediated cytoskeletal rearrangements regulate integrin function in mouse platelets

“…13,21 Similarly, Pfn1 fl/fl-Pf4Cre platelets showed severely delayed and less effective clot retraction ( Figure 1H), further reflecting impaired integrin function and actin dynamics. 10,13 Because Pfn1 is an effector of the RhoA/ Rho-associated kinase (ROCK) pathway, these findings are in agreement with reports on RhoA-deficient platelets that show abolished clot retraction.…”

“…Animals, flow cytometry, aggregation, immunostaining, adhesion under flow, clot retraction, immunoblotting, tail bleeding assay, in vivo model, Ca 21 measurements, atomic force microscopy, and statistical analyses are described in the supplemental Methods. 13 Animal studies were approved by the district government of Lower Franconia (Bezirksregierung Unterfranken).…”

“…Figure 1K-L). This was most likely due to impaired actin dynamics in the absence of Pfn1, 13 because inhibition of activation-dependent actinpolymerization was shown to increase store-operated Ca 21 entry. 24 Besides Ca 21 signaling, Tln1 recruitment to b-integrin tails represents a key step in integrin activation.…”

Proteomics analysis of bladder cancer invasion: Targeting EIF3D for therapeutic intervention

“…The shRNA sequences targeting EIF3D and RHEB were 5′-CCGGCCTCAGACATACTCCATAGATCTCGAGATCTATGGAGTATGTCTGAGGTTTTTG-3′, respectively. A scrambled shRNA (shscramble) was used as control, as previously described [ 119 ]. For the lentivirus production, a four plasmid system was used for the transient transfection of 293T cells as previously described [ 120 , 121 ], followed by concentration with Amicon Ultra Centrifugal Filters-100K Units (Merck Millipore).…”

A balanced level of profilin-1 promotes stemness and tumor-initiating potential of breast cancer cells

“…Interestingly, a recent study reported downregulation of cell-surface integrin b1 expression and Wnt signaling in a bladder cancer cell line upon knockdown of Pfn1 expression. 28 While this may suggest apparent contradictory effects of Pfn1 knockdown between breast and bladder cancer cells, because of differences in experimental parameters between the 2 studies (e.g., surface protein expression vs mRNA level of integrin b1) it is difficult to reconcile these results. Cell-specific differences in the results will not be totally surprising given that Pfn1 has context-specific effects in normal vs cancer cells and even between different cancer cell lines.…”