ABSTRACT Kindlin-2 is a cytoskeletal adapter protein that is present in many different cell types. By virtue of its interaction with multiple binding partners, Kindlin-2 intercalates into

numerous signaling pathways and cytoskeletal nodes. A specific interaction of Kindlin-2 that is of paramount importance in many cellular responses is its direct binding to the cytoplasmic

tails of integrins, an interaction that controls many of the adhesive, migratory and signaling responses mediated by members of the integrin family of cell-surface heterodimers. Kindlin-2 is

highly expressed in many cancers and is particularly prominent in prostate cancer cells. CRISPR/cas9 was used as a primary approach to knockout expression of Kindlin-2 in both

was markedly suppressed. These observations indicate that Kindlin-2 regulates hallmark features of prostate cancer cells. In androgen expressing cells, testosterone-stimulated adhesion was

Kindlin-2-dependent. Furthermore, tumor growth of a prostate cancer cell line lacking Kindlin-2 and implanted into the prostate gland of immunocompromised mice was markedly blunted and was

associated with suppression of angiogenesis in the developing tumor. These results establish a key role of Kindlin-2 in prostate cancer progression and suggest that Kindlin-2 represents an

interesting therapeutic target for treatment of prostate cancer. SIMILAR CONTENT BEING VIEWED BY OTHERS EPHA2 REGULATES VASCULAR PERMEABILITY AND PROSTATE CANCER METASTASIS VIA MODULATION OF

CELL JUNCTION PROTEIN PHOSPHORYLATION Article Open access 07 November 2024 THE NOGO RECEPTOR NGR2, A NOVEL ΑVΒ3 INTEGRIN EFFECTOR, INDUCES NEUROENDOCRINE DIFFERENTIATION IN PROSTATE CANCER

Article Open access 07 November 2022 PLECTIN IS A REGULATOR OF PROSTATE CANCER GROWTH AND METASTASIS Article 20 November 2020 INTRODUCTION Prostate cancer (PCa) is the second leading cause

of cancer deaths in men. With early detection, the 5-years survival rate of patients PCa is more than 98%, reflecting the success of various therapeutic modalities, including hormone

ablation1. However, some PCa tumors become resistant to androgen ablation leading to metastatic castration-resistant prostate cancer (mCRPCa)2. The neuroendocrine subtype of PCa, which is

characterized by expression of neuron-specific proteins, develops from subsets of mCRPCa cells, usually metastasizes, and has life expectancies of less than 1 year. In general, when mCRPCa

occurs, 5-years survivals drop to ~ 30%2. Major steps in mCRPCa involve changes in adhesive, metabolic and signaling properties of PCa cells, allowing them to thrive in local and distant

environmental niches. Kindlin-2 (K2), also referred to by its gene name as FERMT2, is a cytoskeletal adapter protein. By virtue of its capacity to engage, either directly or indirectly, with

many different intracellular binding partners, K2 connects with and influences multiple signaling pathways and cytoskeletal nodes. Like the other two kindlins in mammalian cells, Kindlin-1

(FERMT1) and Kindlin-3 (FERMT3), K2 consists of a FERM domain composed of the typical F1, F2 and F3 subdomains3,4,5.Distinguishing the kindlins from other FERM domain containing proteins is

the insertion of a lipid binding PH domain into the F2 subdomain. K2 is the major kindlin of endothelial cells, smooth muscle cells and fibroblasts6. Deficiency of K2 has not been reported

in humans, but its absence in mice and fish is embryonically lethal7,8. Postnatal knockout of K2 (K2KO) leads to heart and vascular defects9,10. We and many other groups have shown that K2

is highly expressed in cancer11,12,13, but there is only limited information on the roles of K2 in PCa cells14. Our study was undertaken to provide such information. The most

well-established functions of K2 emanate from its capacity to regulate activation of integrins, the transition of these α/β heterodimeric cell-surface receptors from a low affinity to a high

affinity/avidity state for their recognition of an extensive repertoire of cognate ligands15. This function of K2 depends on its binding to the cytoplasmic tails of integrin β subunits

utilizing its K2Q614W615 motif in its C-terminal F3 subdomain for integrin recognition. Expression of wild-type (WT) K2 or the K2Q614W615 mutant in cells in which K2 has been knocked down

provides an effective strategy to dissect which oncogenic responses of PCa cells are dependent on K2 binding to integrins15,16,17,18. Integrin functions are often dysregulated in cancer

cells19,20,21, and elevated K2 levels have been documented in many different cancers12,13,22. In PCa, K2 mRNA level was identified as among the top 3% genes upregulated in an oncomine data

set14, and K2 protein expression was significantly enhanced with PCa progression (https://www.proteinatlas.org/). These correlations have led us to examine the effects of K2 knockdown on

selected hallmark responses of transformed PCa cancer cells in vitro, ranging from altered adhesion, spreading and migration, and on the growth of prostate tumors in vivo_._ RESULTS In our

studies of K2 in PCa, we have used three well-established PCa cell lines: LNCaP (androgen dependent), DU145, and PC3 (both androgen independent). Each of these PCa cell lines express K2

(Fig. 1A). The presence of integrins in these cells as reflected by Western blotting of the integrin β3 subunit suggested the possibility that K2 might regulate functional responses of these

PCa cells. Accordingly, CRISPR/cas9 was used to reduce K2 levels (K2KO) in each of these cell lines (Fig. 1A) and was found to be very effective (more than > 98% in PC3 and DU145 and

more than 70% in LNCaP), Integrin β1 and β3 subunit expression was not altered as shown by Western blots (Fig. 1A). By flow cytometry, cell-surface expression levels of integrin β1 (Fig. 1B)

and β3 subunits (Fig. 1C) were not suppressed by K2KO. While K2 levels were greatly reduced by the CRIPSR/Cas9 approach, K1 and K3 levels in these cell lines were not altered as determined

by Western blotting with specific antibodies (not shown). EFFECTS OF K2 KNOCKDOWN ON THE ADHESIVE PROPERTIES OF PCA CELLS A characteristic of transformed cells is altered adhesive

properties, which often reflects increased integrin expression and/or function21. We compared the adhesion of the three parental and derivative K2KO cells (CRISPR/Cas9 cells) for their

ability to adhere to fibrinogen (FIB), a ligand for integrin αvβ3, or fibronectin (FN), a ligand for integrin α5β1. This response was blunted significantly by K2KO in each of the cell lines

(Fig. 2A–C). Spreading on fibronectin (FN) was also significantly inhibited by K2KO for all three cell lines at 30 min (Fig. 2D); but, as shown for DU145 cells, was similar in the K2KO and

the parental cells at 2 h. Inhibition of cell spreading by the K2-KO cells on FN, was accompanied by changes in the distribution of vinculin, a focal adhesion marker, Vinculin distribution

was visibly different at 30 min, with fewer less-organized focal adhesions in K2KO cells (Fig. S1A), focal adhesions indicated with arrows). Vinculin distribution was also altered at 2 h,

with majority of the staining remaining in the cytoplasm and very few focal adhesions were evident (Fig. S1B) even though, spreading of the attached cells on FN at the 2 h time point had

reached similar levels as parental cells, indicating that there was not a general paralysis of cytoskeletal dependent responses by K2KO. We sought to determine whether the effects of K2

knockdown on adhesion was a consequence of its regulation of integrin function. Accordingly, a rescue strategy was implemented in which PC3 cells with K2KO were transduced with lentiviral

particles containing either wild-type (WT) K2 or K2Q614W615/AA mutant. The two K2 forms encoded for an EGFP-tag at their C-terminus, and Western blotting was used to detect and monitor

similarity in expression levels of the two K2 forms. K2KO cells using CRISPR/Cas9 failed to express either K2 using this transfection strategy. Thus, we used cells in which K2 was knocked

down with shRNA. Two different shRNAs as well as a non-targeting control shRNA were used. As shown in Fig. 4A, the two targeted shRNAs reduced K2 levels by ~ 80%, whereas the non-targeted

control shRNA had no effect of K2 levels (Fig. 3A). The two targeted shRNAs blunted adhesion of the parental PC3 cells as was observed with the CRISPR/Cas9 knock-down, and the non-targeting

shRNA had no significant effect on adhesion (Fig. 3B). WT K2 rescued the adhesion for both K2-targeting shRNAs. In contrast, the K2Q614W615/AA mutant failed to rescue the adhesive response

(Fig. 3B). Thus, the role of K2 in PCa cell adhesion requires its binding to integrins. EFFECTS OF K2 KNOCKDOWN ON MIGRATION OF PCA CELLS Migration is a complex adhesive response, requiring

not only adhesion but also de-adhesion, redistribution of cell-surface receptors and reorganization of the cytoskeleton. We compared the migration of the parental and K2KO cells across a FN

substrate. The effects of K2KO on the migration response in PC and Du145 cells was observed but was less extensive than on adhesion alone (Fig. 4A,B). EFFECT OF K2 KNOCKDOWN ON

ANCHORAGE-INDEPENDENT GROWTH Colony formation in soft agar requires anchorage independent growth, is a hallmark of cancer23,24 and it is regarded as one of the most robust assays for

malignant transformation. As shown in Fig. 5A, colony formation by LNCaP cells was markedly suppressed by K2KO. This effect was also pronounced in the other two PCa cell lines (Fig. 5B).

Thus, K2 contributed to this key property of transformed cells. To determine whether loss of K2 expression may exert its effects by altering the viability of prostate cancer cell lines, we

performed flow cytometry analyses to assess for changes in expression of Annexin V, an established marker of apoptosis. While loss of K2 in LnCAP cells showed no detectable change in Annexin

V expression (Fig. 5C, Left panel), both DU154 and PC3 deficient in K2 showed noticeable increase in Annexin V expression, which is indicative of activation of apoptosis in these two cell

lines as a result of K2 loss. (Fig. 5C, middle and right panels. respectively). This effect is consistent with our previously reported data25. EFFECT OF K2 KNOCKDOWN ON TUMORSPHERE INVASION

To mimic the invasive behavior of cancer cells in tumor microenvironment, we performed 3D-tumorsphere invasion of extracellular matrices. K2-deficient PC3 cells were unable to invade ECM

containing Matrigel (Fig. 5D,E). EFFECT OF K2 KNOCKOUT ON ANDROGEN-INDUCED ADHESIVE RESPONSES Particularly relevant to PCa is the response to androgen stimulation. The androgen-dependent

LNCaP cells and androgen-independent PC3 cells were grown in androgen-free FCS for 24 h, and their adhesive responses induced by testosterone (400 nM) were assessed (Fig. 6A,B). Testosterone

enhanced adhesion of LNCaP cells to fibrinogen (FIB), an integrin αvβ3 ligand, by 2.5-fold and to FN, an α5β1 by twofold (Fig. 6A). Testosterone failed to do so in PC3 cells (Fig. 6B). This

testosterone-dependent enhancement of LNCaP adhesion to these integrin ligands was mediated by androgen receptor (AR) engagement since two AR antagonists, bicalutamide and flutamide,

completely inhibited the effect of testosterone on adhesion (Fig. 6A). In contrast, these AR antagonists did not have any effect on PC3 adhesion as their response to testosterone (Fig. 6B)

was negligible, consistent with the extremely low levels of AR (Table 1). Also, AR antagonists did not abrogate integrin function in any of PCa cells as their adhesion in the absence of

testosterone was not changed by these antagonists. Of note, K2KO with CRISPR/Cas 9 in LNCaP cells suppressed their adhesion to FIB at all 3 doses of the hormone tested (Fig. 6C), suggesting

the importance of K2 in this androgen-dependent response. We excluded some of the most obvious explanations for these dramatic effects: by flow cytometry, surface expression of α5β1 and αvβ3

was not changed by K2KO or by testosterone (Table 1). AR expression, as measured with rabbit polyclonal antibodies, was enhanced by 25% by testosterone (400 nM) in LNCaP cells but was not

significantly different in parental vs K2KO cells, suggesting that K2 does not play any role in regulation of AR expression (Table 1). EFFECT OF K2 KNOCKOUT ON PROSTATE TUMOR GROWTH IN VIVO

Based on the profound effects of K2KO on the properties of the PCa cell lines, we sought to determine if K2KO altered tumor growth in vivo. Two groups of 5 mice were given an intraprostate

injection of 1 × 106 WT PC3 cells or K2KO PC3 cells. A high concentration of cells was used to determine if a dramatic difference due to loss of K2 on tumor growth could be detected. In

fact, the results shown in Fig. 7A were indeed significant (_p_ = 0.037) as K2KO suppressed tumor growth as monitored by IVIS. The experiment was terminated when the size of tumors from the

parental cells reached a predetermined endpoint (17 mm). The tumors were then excised from both WT and K2KO PC3 bearing mice and sectioned and then immunostaining for vWF as a blood vessel

marker to assess angiogenesis. As shown in Fig. 7B, angiogenesis was markedly suppressed in the tumor derived from the K2KO PC3 cells as compared to the parental cells, as detected with vWF

staining. Quantification of vessel area with Image J, the average blood vessel area per tumor section was 14,827.4 µm2 ±  1658.7 (SEM) for parental PC3 cells versus 5746.4 µm2 ± 833.1 (SEM)

for K2KO cells. CA9 was stained as a marker for hypoxia26, and no difference was noted between control and K2-KO tumors (Fig. 7C,D). Mechanistically, when tumors were also stained for VEGF

(Fig. 7E,F) and phosphorylated VEGFR2 (Fig. 7G,H), the expression of these mediators of angiogenetic responses were suppressed in the K2KO PC3 tumors. These observations are consistent with

our previous findings in breast cancer tumors with K2KO in vivo27. DISCUSSION K2 shows enhanced expression in many solid tumors12,13,21. In comparing K2 mRNA levels across several different

solid tumors, K2 was markedly elevated in PCa and was in the top 3% of elevated genes14. Furthermore, K2 protein levels were found to increase as PCa progressed from early to advanced

prostate adenocarcinoma (https://www.proteinatlas.org/). Nevertheless, there is limited information available on how K2 influences PCa cell adhesion, spreading and tumor development in vivo.

The influence of K2 on transformed cell responses controlled by integrins is to be expected in view of the important regulatory role of kindlins in integrin function. Thus, the capacity of

K2 to regulate integrin-mediated adhesive responses of PCa cells, both androgen-and androgen-independent PCa cells, including adhesion and migration, is consistent with the known functions

of K2. The similarity in the effects was noted with two different methods to reduce K2 levels, CRISPR/Cas9 and shRNA K2 knockdown, minimizes the likelihood of off-target effects of the

manipulation used. The influence of K2 on anchorage independent growth is not surprising in view of the established role of integrins in such growth arising from their control of growth

factors and growth factor receptors and/or intracellular signaling pathways that control cellular growth and division28. These results are also consistent with studies showing that

inactivation of K2 enhanced the susceptibility of prostate cancer cells to apoptosis and cell death induced by docetaxel, which was traced to inhibition of β1-integrin function25. Together,

the effects of K2 in these responses establish its critical role in hallmark responses of prostate cancer cells. Of note, talin-1 which collaborates with K2 in integrin activation18 also is

elevated in PCa29 the response of the AR expressing cells to hormone was K2 dependent. The signaling pathways by which K2 regulates the adhesive responses to testosterone could involve an

ER-stress response30. K2KO in PC3 cells had a dramatic effect on tumor growth in the prostate in mice. The substantial suppression of tumor angiogenesis is likely to be a major contributing

factor to the inhibition of tumor growth. This supposition is consistent with the effect of K2KO in breast tumor development31 and on the effects of K2 reduction on a mouse prostate tumor

(RM1) growth and vascularization implanted into Matrigel plugs in mice as well as other models of vascular permeability and angiogenesis32,33. If such profound effects are observed with

other PCa cells and other prostate cancer models, reduction of K2 levels might be considered as a potential therapeutic target for prostate cancer as well as other cancers. This possibility

could be achieved by selectively targeting K2 suppressors to tumor cells or by limiting the extent of suppression of K2 expression. In this regard, it should be noted that 50% reduction of

K2 expression in K2+/− mice was without overt deleterious effects32. Thus, partial reduction in K2 might be sufficient to limit cancer progression. METHODS ANTIBODIES AND REAGENTS Mouse mAb

against K2 (clone 3A3) was from Millipore Sigma, rabbit mAb against actin (D18C11) was from Cell Signaling Technology, mouse mAb against vinculin was from Millipore Sigma; mouse anti human

aVb3-AF488 (clone LM609) was from Milipore Sigma; mouse anti CA9 mAb was from Abcam; mouse anti EGFP mAb (B2) was from Santa Cruz Biotechnology; rabbit anti VEGF-R mAb was from Cell

Signalling Technology; anti-human mouse mAb against β1-integrin PE-conjugated was from BD Pharmingen; rabbit anti- human AR-AF488 was from Millipore Sigma; Alexa-coupled secondary

antibodies, and Alexa-coupled phalloidins were from Thermo Fisher Scientific; rabbit polyclonal antibodies against phospho-VEGF R2 (Tyr1054, Tyr1059) were from Invitrogen; rabbit polyclonal

antibodies against vWf were from Agilent; horseradish peroxidase–conjugated secondary antibodies were from Bio-Rad; bovine fibronectin was from Milipore Sigma, human fibrinogen was from

Milipore Sigma, testosterone was from Milipore Sigma; ECL reagent was from Roche, RPMI, DMEM/F12, penicillin/streptomycin, and l-glutamine were from the Media Lab (Cleveland Clinic). CELL

LINES AND CULTURE Human PC3, DU145 and LNCaP cells were purchased from American Type Culture Collection (ATCC). DU145 and LNCaP cells were grown in RPMI with 10% fetal calf serum (FCS), 100

units penicillin/ml and 100 μg streptomycin/ml. PC3 cells were maintained in Dulbecco’s modified Eagle’s medium F12 supplemented with 10% FBS, 100 units of penicillin/ml, 100 μg of

streptomycin. Although cell line authentication was not explicitly conducted, we relied on the manufacturer’s quality control assurances. Periodic testing for Mycoplasma contamination was

performed every 9–12 months. All cells were cultured at early passages (no more than 15), and each culture was passaged no more than five times before introducing a fresh vial. MANIPULATION

OF K2 EXPRESSION IN PCA CELLS K2-KO by CRISPR/Cas9: K2 -deficient cells were generated through electroporation of cancer cells with a ribonucleoprotein mixture of guide RNAs (sgRNA) and Cas9

(Synthego), following the manufacturer’s instructions. A pool of two verified sgRNAs (sgRNA-1:5′-AGGCGTGATGCTTAAGCTGG-3′ and sgRNA-2:5′-GGTATACTTGCTGTCAGTCA-3′) was used for human FERMT2

gene (Synthego), with scrambled sgRNAs serving as a negative control. Western blot (WB) analysis validated efficient and stable knockout (KO). In cases where knockdown efficiency was below

80%, a second round of sgRNA delivery was implemented. No antibiotic selection was required, as the knockdown efficiency was sustained throughout the cells’ utilization. K2 downregulation

with shRNA: shRNA-mediated knockdown of K-2 was performed as previously described25. RE-EXPRESSION OF EGFP-TAGGED K2WT AND K2Q614W615/AA IN K2KO PCA CELLS The PCa cells were treated with

lentiviral vectors as described34 expressing EGFP-tagged K2 (WT), Q614W615/AA, or EGFP only as described34. 48 h post-transduction, the cells were used in adhesion experiments. COLONY

FORMATION ASSAY Cells were seeded into 6-well plates (300 cells per well) and cultured for 10 days. Fresh medium was changed every 3 days. At the end of the experiment, the forming colonies

were washed with PBS, fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min, and stained with 0.25% crystal violet solution. Image acquisition of the 6-well plate and

quantification of clones were performed using the ChemiDoc MP Imaging system (Bio-Rad) and ImageJ software. 3D-TUMORSPHERE AND INVASION ASSAYS Cells (2500 cells per well) were seeded into a

96-well ultralow attachment (ULA) plate and monitored for 12 days, as described previously35. Invasion assays involved supplementing tumorspheres cultures with Matrigel, and invasion was

monitored for 10 additional days. Images were captured and quantified using ImageJ software35. ANDROGEN STIMULATION The PCa cells at 70–80% confluency were incubated overnight in respective

RPMI or DMEM F-12 media containing 2% hormone-free (charcoal-stripped) FBS (Thermofisher Scientific). Next day, testosterone was added at 100 or 400 nM and the cells were incubated for 24 h

before commencement of experiments. In samples pretreated with AR antagonists, Bicalutamide (5 µM, Tocris Biotechne) or Flutamide (30 µM, Tocris Biotechne) were added 1 h before treatment

with testosterone. ADHESION ASSAYS 96-well plates were coated with FN (100 µl/well of 10 µg/ml) or FIB (100 µl/well of 5 µg/ml) in PBS overnight at 4 °C followed by 1 h at 37 °C. The plates

were post-coated with 200 µl/well of 0.5% polyvinylpyrrolidone (PVP) in PBS for 1 h at 37 °C. The PCa cells either untreated or upon various treatments were added at 50,000 cells/well in

HBSS buffer, containing 0.1% BSA, 1 mM CaCl2, 1 mM MgCl2 and incubated for 40 min at 37 °C. Nonadherent cells were aspirated and the plates were washed twice with PBS. The numbers of

adherent cells were quantified using the CyQuant Cell Proliferation Assay kit according to manufacturer’s instructions. MIGRATION ASSAYS PCa cell migration was assessed in serum-free DMEM

F-12 medium using Costar 24-transwell plates with 8-μm pore polycarbonate filters (Corning, Corning, NY) with immobilized FN or FIB (10 µg/ml) on their lower surface. PC3 or DU145 cells (1 ×

 105) were added to the upper chambers and incubated in a humidified incubator for 16 h at 37 °C and 5% CO2. Assays were stopped by removing the upper wells and wiping the inside of upper

wells with a cotton swab to remove nonmigrated cells. The migrated cells were quantitated using the Cyquant Cell Proliferation Kit. CELL SPREADING PC3, DU145 and LNCaP cells were plated onto

ligand-coated glass slides for the times indicated, fixed with 4% PFA, permeabilized with 0.1% Triton X-100, blocked in horse serum, and stained with Alexa-648 phalloidin to visualize actin

and vinculin antibody, followed by anti-mouse goat Alexa 488-conjugated secondary antibody to visualize focal adhesions. Stained cells were visualized with a 40 × oil objective using a

Leica TCS-NT laser scanning confocal microscope (Imaging Core; Cleveland Clinic). Images were collected using Leica Confocal Software (version 2.5 Build 1227). TUMOR TISSUE IMMUNOSTAINING

Tumor were collected after 40 days after tumor implantation, snap-frozen in optimal cutting temperature medium (Sakura Finetek), and 8-μm sections were prepared. Tumor sections were stained

with antibodies indicated and stained sections were analyzed using fluorescent imaging microscopy (Leica) and ImageJ software. FLOW CYTOMETRY The PCa cells, upon respective treatments, were

incubated (0.5 × 106 in 100 µl of HBSS buffer containing 0.1% BSA, 1 mM CaCl2, 1 mM MgCl2) with the following antibodies: anti- human αVβ3-AF488 (clone LM609, Millipore Sigma) , anti-human

CD29-PE (BD Pharmigen) anti-human CD61-PE (BD Pharmigen), anti-human Annexin V-PE (BD Pharmigen) or anti-human AR-AF488 (Millipore Sigma) at 0.5–1 µg/sample for 30 min on ice. The cells were

permeabilized using the Permeabilization buffer (eBioscience) for 5 min at RT before staining for AR. The cells were washed 2 × with 400 µl of the HBSS buffer and fixed with 1% PFA in HBSS

buffer. The samples were measured using BD FACSymphony A1 Cell Analyzer and analyzed with the FlowJo v10.8.1 software. PRIMARY TUMOR GROWTH AND BIOLUMINESCENCE IMAGING Control (i.e.,

nonsilencing shRNA) and K2-deficient luciferase-expressing PC3 cells were engrafted (106 cells/mouse) into the prostate of male NSG mice. The growth and metastasis of primary tumors were

quantified biweekly bioluminescent imaging of tumor-bearing animals on a Xenogen IVIS-200 (Caliper Life Sciences, Hopkinton, MA, USA) in the Imaging Research Core (Cleveland Clinic Lerner

Research Institute, Cleveland, OH, USA). Bioluminescence imaging was performed 30 min after inoculation (T0) and biweekly thereafter. ANIMAL STUDIES Animal experiments using mice were

performed in accordance with recommendations in Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and conducted under a protocol approved by Cleveland

Clinic Institutional Animal Care and Use Committee. Animal experiments using mice were performed in compliance with the ARRIVE guidelines. Studies were supported in part by the Case

Comprehensive Cancer Center Athymic Animal and Preclinical Therapeutics Shared Resource and NCI core grant 5 P30 CA043703-32. DATA AVAILABILITY The datasets used and/or analyzed during the

current study are available from the corresponding author on reasonable request. REFERENCES * Desai, K., McManus, J. M. & Sharifi, N. Hormonal Therapy for Prostate Cancer. _Endocr. Rev._

42, 354–373. https://doi.org/10.1210/endrev/bnab002 (2021). Article  PubMed  PubMed Central  Google Scholar  * Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2016. _CA

Cancer J. Clin._ 66, 7–30. https://doi.org/10.3322/caac.21332 (2016). Article  PubMed  Google Scholar  * Moser, M., Legate, K. R., Zent, R. & Fassler, R. The tail of integrins, talin,

and kindlins. _Science_ 324, 895–899. https://doi.org/10.1126/science.1163865 (2009). Article  ADS  PubMed  CAS  Google Scholar  * Meller, J. _et al._ Emergence and subsequent functional

specialization of kindlins during evolution of cell adhesiveness. _Mol. Biol. Cell_ 26, 786–796. https://doi.org/10.1091/mbc.E14-08-1294 (2015). Article  PubMed  PubMed Central  CAS  Google

Scholar  * Plow, E. F. & Qin, J. The Kindlin family of adapter proteins. _Circ. Res._ 124, 202–204. https://doi.org/10.1161/CIRCRESAHA.118.314362 (2019). Article  PubMed  PubMed Central

  CAS  Google Scholar  * Karakose, E., Schiller, H. B. & Fassler, R. The kindlins at a glance. _J. Cell Sci._ 123, 2353–2356. https://doi.org/10.1242/jcs.064600 (2010). Article  PubMed 

CAS  Google Scholar  * Montanez, E. _et al._ Kindlin-2 controls bidirectional signaling of integrins. _Genes Dev._ 22, 1325–1330 (2008). Article  PubMed  PubMed Central  CAS  Google Scholar

  * Dowling, J. J. _et al._ Kindlin-2 is an essential component of intercalated discs and is required for vertebrate cardiac structure and function. _Circ. Res._ 102, 423–431.

https://doi.org/10.1161/CIRCRESAHA.107.161489 (2008). Article  PubMed  CAS  Google Scholar  * Zhang, Z. _et al._ Postnatal Loss of Kindlin-2 Leads to Progressive Heart Failure. _Circ. Heart

Fail._ 9, e003129. https://doi.org/10.1161/CIRCHEARTFAILURE.116.003129 (2016). Article  PubMed  CAS  Google Scholar  * Zhang, Z. _et al._ Kindlin-2 is essential for preserving integrity of

the developing heart and preventing ventricular rupture. _Circulation_ 139, 1554–1556. https://doi.org/10.1161/CIRCULATIONAHA.118.038383 (2019). Article  PubMed  PubMed Central  Google

Scholar  * Wei, C. Y. _et al._ Elevated kindlin-2 promotes tumour progression and angiogenesis through the mTOR/VEGFA pathway in melanoma. _Aging (Albany NY)_ 11, 6273–6285.

https://doi.org/10.18632/aging.102187 (2019). Article  PubMed  CAS  Google Scholar  * Plow, E. F., Das, M., Bialkowska, K. & Sossey-Alaoui, K. Of kindlins and cancer. _Discoveries

(Craiova)_ 20, 30. https://doi.org/10.15190/d.2016.6 (2016). Article  Google Scholar  * Wang, W., Kansakar, U., Markovic, V. & Sossey-Alaoui, K. Role of Kindlin-2 in cancer progression

and metastasis. _Ann. Transl. Med._ 8, 901. https://doi.org/10.21037/atm.2020.03.64 (2020). Article  PubMed  PubMed Central  CAS  Google Scholar  * Nanni, S. _et al._ Epithelial-restricted

gene profile of primary cultures from human prostate tumors: A molecular approach to predict clinical behavior of prostate cancer. _Mol. Cancer Res._ 4, 79–92.

https://doi.org/10.1158/1541-7786.MCR-05-0098 (2006). Article  PubMed  CAS  Google Scholar  * Ma, Y. Q., Qin, J., Wu, C. & Plow, E. F. Kindlin-2 (Mig-2): A co-activator of beta3

integrins. _J. Cell Biol._ 181, 439–446 (2008). Article  PubMed  PubMed Central  CAS  Google Scholar  * Shi, X. _et al._ The MIG-2/integrin interaction strengthens cell-matrix adhesion and

modulates cell motility. _J. Biol. Chem._ 282, 20455–20466 (2007). Article  PubMed  CAS  Google Scholar  * Pluskota, E. _et al._ Kindlin-2 regulates hemostasis by controlling endothelial

cell-surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism. _Blood_ 122, 2491–2499. https://doi.org/10.1182/blood-2013-04-497669 (2013). Article  PubMed  PubMed

Central  CAS  Google Scholar  * Lu, F. _et al._ Mechanism of integrin activation by talin and its cooperation with kindlin. _Nat. Commun._ 13, 2362.

https://doi.org/10.1038/s41467-022-30117-w (2022). Article  ADS  PubMed  PubMed Central  CAS  Google Scholar  * Varner, J. A. & Cheresh, D. A. Integrins and cancer. _Curr. Opin. Cell

Biol._ 8, 724–730 (1996). Article  PubMed  CAS  Google Scholar  * Weis, S. M. & Cheresh, D. A. alphaV integrins in angiogenesis and cancer. _Cold Spring Harb. Perspect. Med._ 1, a006478.

https://doi.org/10.1101/cshperspect.a006478 (2011). Article  PubMed  PubMed Central  CAS  Google Scholar  * Hamidi, H. & Ivaska, J. Every step of the way: Integrins in cancer

progression and metastasis. _Nat. Rev. Cancer_ 18, 533–548. https://doi.org/10.1038/s41568-018-0038-z (2018). Article  PubMed  PubMed Central  CAS  Google Scholar  * Augoff, K. _et al._

miR-31 is a broad regulator of beta1-integrin expression and function in cancer cells. _Mol. Cancer Res._ 9, 1500–1508. https://doi.org/10.1158/1541-7786.MCR-11-0311 (2011). Article  PubMed

  PubMed Central  CAS  Google Scholar  * Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. _Cell_ 100, 57–70. https://doi.org/10.1016/s0092-8674(00)81683-9 (2000). Article  PubMed 

CAS  Google Scholar  * Hanahan, D. Hallmarks of cancer: New dimensions. _Cancer Discov._ 12, 31–46. https://doi.org/10.1158/2159-8290.CD-21-1059 (2022). Article  PubMed  CAS  Google Scholar

  * Sossey-Alaoui, K. & Plow, E. F. miR-138-mediated regulation of KINDLIN-2 expression modulates sensitivity to chemotherapeutics. _Mol. Cancer Res._ 14, 228–238.

https://doi.org/10.1158/1541-7786.MCR-15-0299 (2016). Article  PubMed  CAS  Google Scholar  * Giatromanolaki, A. _et al._ Expression of hypoxia-inducible carbonic anhydrase-9 relates to

angiogenic pathways and independently to poor outcome in non-small cell lung cancer. _Cancer Res._ 61, 7992–7998 (2001). PubMed  CAS  Google Scholar  * Sossey-Alaoui, K. _et al._ Kindlin-2

regulates the growth of breast cancer tumors by activating CSF-1-mediated macrophage infiltration. _Cancer Res._ 77, 5129–5141. https://doi.org/10.1158/0008-5472.CAN-16-2337 (2017). Article

  PubMed  PubMed Central  CAS  Google Scholar  * Renshaw, M. W., Price, L. S. & Schwartz, M. A. Focal adhesion kinase mediates the integrin signaling requirement for growth factor

activation of MAP kinase. _J. Cell Biol._ 147, 611–618 (1999). Article  PubMed  PubMed Central  CAS  Google Scholar  * Desiniotis, A. & Kyprianou, N. Significance of talin in cancer

progression and metastasis. _Int. Rev. Cell Mol. Biol._ 289, 117–147. https://doi.org/10.1016/B978-0-12-386039-2.00004-3 (2011). Article  PubMed  PubMed Central  CAS  Google Scholar  *

Macke, A. J. _et al._ Targeting the ATF6-mediated ER stress response and autophagy blocks integrin-driven prostate cancer progression. _Mol. Cancer Res._ 21, 958–974.

https://doi.org/10.1158/1541-7786.MCR-23-0108 (2023). Article  PubMed  PubMed Central  CAS  Google Scholar  * Sossey-Alaoui, K., Pluskota, E., Szpak, D., Schiemann, W. P. & Plow, E. F.

The Kindlin-2 regulation of epithelial-to-mesenchymal transition in breast cancer metastasis is mediated through miR-200b. _Sci. Rep._ 8, 7360. https://doi.org/10.1038/s41598-018-25373-0

(2018). Article  ADS  PubMed  PubMed Central  CAS  Google Scholar  * Pluskota, E. _et al._ The integrin coactivator kindlin-2 plays a critical role in angiogenesis in mice and zebrafish.

_Blood_ 117, 4978–4987. https://doi.org/10.1182/blood-2010-11-321182 (2011). Article  PubMed  PubMed Central  CAS  Google Scholar  * Pluskota, E. _et al._ Kindlin-2 interacts with

endothelial adherens junctions to support vascular barrier integrity. _J. Physiol.-Lond._ 595, 6443–6462. https://doi.org/10.1113/Jp274380 (2017). Article  PubMed  PubMed Central  CAS 

Google Scholar  * Sossey-Alaoui, K., Pluskota, E., Szpak, D. & Plow, E. F. The Kindlin2-p53-SerpinB2 signaling axis is required for cellular senescence in breast cancer. _Cell Death

Dis._ 10, 539. https://doi.org/10.1038/s41419-019-1774-z (2019). Article  PubMed  PubMed Central  CAS  Google Scholar  * Rana, P. S. _et al._ The WAVE2/miR-29/integrin-beta1 oncogenic

signaling axis promotes tumor growth and metastasis in triple-negative breast cancer. _Cancer Res. Commun._ 3, 160–174. https://doi.org/10.1158/2767-9764.CRC-22-0249 (2023). Article  PubMed

  PubMed Central  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The work at the Cleveland Clinic investigators (KB, EFP and EP) was supported by NHLBI P01HL154811. These

investigators received no financial support from the $14.3M raised for cancer research at the Cleveland Clinic. KSA was supported by R01CA226921 and LRL by DoD W81XWH2210826. AUTHOR

INFORMATION Author notes * These authors contributed equally: Katarzyna Bialkowska and Lamyae El Khalki. AUTHORS AND AFFILIATIONS * Department of Cardiovascular Biology and Metabolic

Sciences, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH, 44139, USA Katarzyna Bialkowska, Elzbieta Pluskota & Edward F. Plow * Department of Medicine, Case

Western Reserve University School of Medicine, Cleveland, OH, USA Lamyae El Khalki, Priyanka S. Rana, Wei Wang & Khalid Sossey-Alaoui * Case Comprehensive Cancer Center, Cleveland, OH,

USA Lamyae El Khalki, Priyanka S. Rana, Wei Wang, Daniel J. Lindner, Yvonne Parker & Khalid Sossey-Alaoui * Department of Pharmacology, Physiology and Cancer Biology, Sidney Kimmel

Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Lucia R. Languino * Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA Dario

C. Altieri * Translational Hematology and Oncology Research, Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA Daniel J. Lindner * Division of Cancer Biology, MetroHealth Medical

Center, Rammelkamp Center for Research, R457, 2500 MetroHealth Drive, Cleveland, OH, 44109, USA Khalid Sossey-Alaoui Authors * Katarzyna Bialkowska View author publications You can also

search for this author inPubMed Google Scholar * Lamyae El Khalki View author publications You can also search for this author inPubMed Google Scholar * Priyanka S. Rana View author

publications You can also search for this author inPubMed Google Scholar * Wei Wang View author publications You can also search for this author inPubMed Google Scholar * Daniel J. Lindner

View author publications You can also search for this author inPubMed Google Scholar * Yvonne Parker View author publications You can also search for this author inPubMed Google Scholar *

Lucia R. Languino View author publications You can also search for this author inPubMed Google Scholar * Dario C. Altieri View author publications You can also search for this author

inPubMed Google Scholar * Elzbieta Pluskota View author publications You can also search for this author inPubMed Google Scholar * Khalid Sossey-Alaoui View author publications You can also

search for this author inPubMed Google Scholar * Edward F. Plow View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.B. performed experiments,

wrote paper and assembled figures; P.R.S. performed experiments and assembled figures; E.P. performed experiments, L.E.K. performed experiments, W.W. generated K2KO cell lines, D.L. and

Y.P. performed in vivo experiments, L.R.L. and D.C.A. reviewed data and edited manuscript, K.S.A. performed experiments and edited manuscript EFP performed experiments, wrote and edited

manuscript. CORRESPONDING AUTHORS Correspondence to Khalid Sossey-Alaoui or Edward F. Plow. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

INFORMATION PUBLISHER'S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY FIGURES. RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits

any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the

Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of

it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material

is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission

directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Bialkowska, K., El Khalki, L., Rana, P.S. _et al._ Role of Kindlin 2 in prostate cancer. _Sci Rep_ 14, 19809 (2024). https://doi.org/10.1038/s41598-024-70202-2 Download citation * Received:

08 April 2024 * Accepted: 12 August 2024 * Published: 27 August 2024 * DOI: https://doi.org/10.1038/s41598-024-70202-2 SHARE THIS ARTICLE Anyone you share the following link with will be

able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing

initiative KEYWORDS * Kindlin-2 * Prostate cancer * Integrins * Tumor progression * Androgen dependence * Tumor angiogenesis