A High-Content, Multiplexed Screen in Human Breast Cancer Cells Identifies Profilin-1 Inducers with Anti-Migratory Activities

Abstract

Profilin-1 (Pfn-1) is a ubiquitously expressed actin-binding protein that is essential for normal cell proliferation and migration. In breast cancer and several other adenocarcinomas, Pfn-1 expression is downregulated when compared to normal tissues. Previous studies from our laboratory have shown that genetically modulating Pfn-1 expression significantly impacts proliferation, migration, and invasion of breast cancer cells in vitro, and mammary tumor growth, dissemination, and metastatic colonization in vivo. Therefore, small molecules that can modulate Pfn-1 expression could have therapeutic potential in the treatment of metastatic breast cancer. The overall goal of this study was to perform a multiplexed phenotypic screen to identify compounds that inhibit cell motility through upregulation of Pfn-1. Screening of a test cassette of 1280 compounds with known biological activities on an Oris™ Pro 384 cell migration platform identified several agents that increased Pfn-1 expression greater than two-fold over vehicle controls and exerted anti-migratory effects in the absence of overt cytotoxicity in MDA-MB-231 human breast cancer cells. Concentration-response confirmation and orthogonal follow-up assays identified two bona fide inducers of Pfn-1, purvalanol and tyrphostin A9, that confirmed in single-cell motility assays and Western blot analyses. SiRNA-mediated knockdown of Pfn-1 abrogated the inhibitory effect of tyrphostin A9 on cell migration, suggesting Pfn-1 is mechanistically linked to tyrphostin A9′s anti-migratory activity. The data illustrate the utility of the high-content cell motility assay to discover novel targeted anti-migratory agents by integrating functional phenotypic analyses with target-specific readouts in a single assay platform.

Facile preparation of a photoactivatable surface on a 96-well plate: a versatile and multiplex cell migration assay platform

“…56 Our results showed the same order value, and therefore it is within acceptable agreement with this report, considering the different cell lines used in these studies. 56 Our results showed the same order value, and therefore it is within acceptable agreement with this report, considering the different cell lines used in these studies.…”

Regulators of actin filament barbed ends at a glance

“…However, whether this property of profilin is physiologically relevant is questionable, as it is generally thought that all profilin is bound to G-actin in cells Xue and Robinson, 2013). High concentrations of profilin (10 to 100 µM), obtained either by microinjection or by overexpression, have however been reported to inhibit cell migration and cause disappearance of filaments in lamellipodia (Cao et al, 1992;Joy et al, 2014;Rotty et al, 2015). In the current view, increasing the cellular concentration of profilin should result in filament disassembly (to give a concentration of the profilin-actin complex of about 45 μM) and thus should enhance local actin-based motile processes rather than inhibit them.…”

“…Profilin also inhibits filament branching in vitro (Machesky et al, 1999;Rotty et al, 2015;Suarez et al, 2015); this inhibition was measured in vitro in conditions where free profilin was present in an excess over profilin-actin, and was proposed to account for the inhibition of cell migration and the disappearance of the lamellipodial filament array at high concentration of profilin (Cao et al, 1992;Joy et al, 2014). However, as discussed above, in vitro and in vivo situations cannot be easily compared.…”

High content analysis in amyotrophic lateral sclerosis

“…The complicated and multi-factorial nature of ALS can definitely affect the success of a single-target based approach and progress towards a therapy with substantive clinical efficacy will most probably call for research strategies that evaluates multi-target approaches 96,97 . The accumulation of protein aggregates, together with excessive cytotoxicity, high level of oxidative stress and inflammation, impaired mitochondria and ER stress 3,98 are all possible targets for designing new therapies. For instance, combining cellular in vitro system with automated microscopy may dramatically increase the speed at which we test new drugs, compounds, or other therapeutic approaches influencing MN survival and morphology.…”