Viktor Todorović

The Chemopreventive Bioflavonoid Apigenin Inhibits Prostate Cancer Cell Motility through the Focal Adhesion Kinase/Src Signaling Mechanism

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratch-wound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of “exaggerated filopodia,” which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness.

Plakoglobin regulates cell motility through Rho- and fibronectin-dependent Src signaling

We previously showed that the cell–cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PG−/− cells onto ECM deposited by PG+/− cells partially restored normal cell morphology and inhibited PG−/− cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PG−/− cells, a substantial decrease in fibronectin (FN) in PG−/− cells stood out. Re-introduction of PG into PG−/− cells restored FN expression, and keratinocyte motility was reversed by plating PG−/− cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PG−/− cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PG−/− cells on ECM deposited by PG+/− keratinocytes. PG−/− cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG−/− keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM–Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.

Fibronectin Expression Determines Skin Cell Motile Behavior

Mouse keratinocytes migrate significantly slower than their human counterparts in vitro on uncoated surfaces. We tested the hypothesis that this is a consequence of differences in the extracellular matrix (ECM) that cells deposit. In support of this, human keratinocyte motility was dramatically reduced when plated onto the ECM of mouse skin cells whereas the latter cells migrated faster when plated onto human keratinocyte ECM. The ECM of mouse and human keratinocytes contained similar levels of the α3 laminin subunit of laminin-332. However, mouse skin cells expressed significantly more fibronectin (FN) than human cells. To assess whether FN is a motility regulator, we utilized siRNA to reduce expression of FN in mouse keratinocytes. The treated mouse keratinocytes moved significantly more rapidly than wild-type mouse skin cells. Moreover, the FN depleted mouse cell ECM supported increased migration of both mouse and human keratinocytes. Furthermore, the motility of human keratinocytes was slowed when plated onto FN-coated substrates or human keratinocyte ECM supplemented with FN in a dose dependent manner. Consistent with these findings, the ECM of α3 integrin-null keratinocytes, which also migrated faster than wild-type cells, was FN deficient. Our results provide evidence that FN is a brake to skin cell migration supported by laminin-332-rich matrices.

Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation

Desmosomal Armadillo family member Pkp3 is established as a coordinator of desmosome and adherens junction assembly and maturation through its physical and functional association with Rap1. It thus functions in a manner distinct from the closely related Pkp2.

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mm ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin β4, and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG’s effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.

Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell-cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ≈ 30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ≈ 2 × larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell-cell and cell-substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis.

Estrogen-dependent sushi domain containing 3 regulates cytoskeleton organization and migration in breast cancer cells

Aromatase inhibitors (AIs) are the standard endocrine therapy for postmenopausal breast cancer; however, currently used biomarkers, such as, estrogen receptor-alpha/progesterone receptor (ERα/PR), predict only slightly more than half of the potential responders to AI treatment. To identify novel markers of AI responsiveness, a genome-wide microarray analysis was performed using primary breast tumor samples from 50 postmenopausal women who later developed metastatic breast cancer. Sushi domain containing 3 (SUSD3) is a significantly differentially expressed gene, with 3.38-fold higher mRNA levels in AI-responsive breast tumors vs non-responders (P<0.001). SUSD3 was highly expressed in ERα-positive breast tumors and treatment with estradiol increased SUSD3 expression in ERα-positive breast cancer cells. Treatment with an antiestrogen or ERα knockdown abolished basal and estradiol-dependent SUSD3 expression. Recruitment of ERα upstream of the transcription start site of SUSD3 was demonstrated by chromatin immunoprecipitation–PCR. Flow cytometric analysis of SUSD3-knockdown cells revealed blunted estradiol effects on progression into S and M phases. SUSD3 was localized to the plasma membrane of breast cancer cells. SUSD3 knockdown decreased the appearance of actin-rich protrusions, stress fibers and large basal focal adhesions, while increasing the presence of cortical actin concomitant with a decrease in Rho and focal adhesion kinase activity. SUSD3-deficient cells demonstrated diminished cell spreading, cell–cell adhesion and motility. In conclusion, SUSD3 is a novel promoter of estrogen-dependent cell proliferation and regulator of cell–cell and cell–substrate interactions and migration in breast cancer. It may serve as a novel predictor of response to endocrine therapy and potential therapeutic target.

Desmosomes and Hemidesmosomes

Desmosomes and hemidesmosomes are adhesive junctions important for maintaining adherence within epithelial tissues – a function reflected in the Greek root desmo , meaning bound. Desmosomes facilitate adhesion between adjacent epithelial cells, whereas hemidesmosomes, named for their ultrastructural resemblance to half a desmosome, mediate adhesion between basal cells of epithelial tissues and the substratum. These junctions are functionally alike in their ability to couple the intermediate filament cytoskeleton to sites of cell–cell or cell–substratum contact at the plasma membrane. However, these organelles differ dramatically in their molecular composition and specialized functions.

Abstract 4221: Plakophilin 3 (Pkp3) increases oral squamous carcinoma cell-cell adhesion while inhibiting cell motility and proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Desmosomal junctions are major contributors to the stability of mechanically stressed tissues including oral epithelium. Plakophilins are a subfamily of Armadillo proteins with a critical role in desmosomal assembly. They have emerged as candidates for a number of regulatory roles both dependent and independent of their function as cell-cell adhesion molecules. These include potential regulation of transcription and translation, as well as control of various signaling cascades. However, there is little known about the biological consequences of their actions. We show here that plakophilin 3 (Pkp3) plays a prominent regulatory role in oral squamous carcinoma cells (OSCC). OSCCs present a great clinical challenge with poor prognosis and no improvement in survival rates for the last 20 years. Using 141 patient tissue samples we demonstrated, by immunoblotting and immunofluorescence, an inverse correlation between Pkp3 and tumor aggressiveness. In addition, >40% of tumor samples completely lost Pkp3 expression. In order to test its role in OSCC cell-cell adhesion, we ablated Pkp3 in the oral cancer cell line SCC9. Pkp3 loss disrupted desmosomal assembly by preventing proper cell border localization of the obligate desmosomal component desmoplakin (DP). Not surprisingly, Pkp3 dependent disruption of desmosomal assembly severely diminished cell-cell adhesion, while at the same time increasing cell motility in a wound healing assay. Interestingly, the activators of cAMP dependent signaling partially rescued the Pkp3 deficient phenotype, demonstrating a role for cAMP in Pkp3 dependent maintenance of oral keratinocyte cell-cell adhesion strength, an important impediment to OSCC growth, motility and invasion. Moving beyond cell-cell adhesion, our preliminary data indicated that Pkp3 down-regulation increases cell cycle progression and proliferation in OSCC cells. The levels of cell cycle inhibitors p21 and p27 were decreased in the absence of Pkp3 whereas the proliferation marker PCNA was increased. Correspondingly, over-expression of Pkp3 in the aggressive OSCC cell line SCC 22B led to an increase in p21 and p27. Both of these results point to Pkp3 acting as a regulator of cell cycle progression, thus we further tested the ability of Pkp3 to regulate cell cycle using flow cytometry. Decreased Pkp3 expression led to an increase in S and G2/M phases of the cell cycle and a concomitant decrease in G0/G1 phase in SCC9 cells indicating an increase in cell cycle progression. Furthermore, a marker of G2 progression, Cyclin B1 is increased in Pkp3 depleted SCC9 cells, indicating that the cells are not experiencing G2/M arrest. In summary, these results support the hypothesis that Pkp3 attenuates OSCC tumor progression towards more aggressive phenotype through both adhesion and cell cycle regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4221. doi:1538-7445.AM2012-4221