Kindlin-2 Regulates the Oncogenic Activities of Integrins and TGF-β In Triple Negative Breast Cancer Progression and Metastasis

Background Kindlin-2, an adaptor protein, is dysregulated in various human cancers, including triple negative breast cancer (TNBC), where it drives tumor progression and metastasis by influencing several cancer hallmarks. One well-established role of Kindlin-2 involves the regulation of integrin signaling, achieved by directly binding to the cytoplasmic tail of the integrin β subunit. In this study, we present novel insights into Kindlin-2’s involvement in stabilizing the β1-Integrin:TGF-β type 1 receptor (TβRI) complexes, acting as a physical bridge that links β1-Integrin to TβRI. The loss of Kindlin-2 results in the degradation of this protein complex, leading to the inhibition of downstream oncogenic pathways. Methods Our methodology encompassed a diverse range of in vitro assays, including CRISPR/Cas9 gene editing, cell migration, 3D tumorsphere formation and invasion, solid binding, co-immunoprecipitation, cell adhesion and spreading assays, as well as western blot and flow cytometry analyses, utilizing MDA-MB-231 and 4T1 TNBC cell lines. Additionally, preclinical in vivo mouse models of TNBC tumor progression and metastasis were employed to substantiate our findings. Results The investigation revealed that the direct interaction between Kindlin-2 and β1-Integrin is mediated through the C-terminal F3 domain of Kindlin-2, while the interaction between Kindlin-2 and TβRI is facilitated through the F2 domain of Kindlin-2. Disruption of this bridge, achieved via CRISPR/Cas9-mediated knockout of Kindlin-2, led to the degradation of β1-Integrin and TβRI, resulting in the inhibition of oncogenic pathways downstream of both proteins, subsequently hindering tumor growth and metastasis. Treatment of Kindlin-2-deficient cells with the proteasome inhibitor MG-132 restored the expression of both β1-Integrin and TβRI. Furthermore, the rescue of Kindlin-2 expression reinstated their oncogenic activities both in vitro and in vivo. Conclusions This study identifies a novel function of Kindlin-2 in stabilizing the β1-Integrin:TβR1 complexes and regulating their downstream oncogenic signaling. The translational implications of these findings are substantial, potentially unveiling new therapeutically targeted pathways crucial for the treatment of TNBC tumors.


Background
Breast Cancer (BC) ranks as the second leading cause of cancer-related deaths among women in the United States, with nearly 300,000 new cases reported annually and over 43,000 lives lost [1].The acquisition of metastatic phenotypes accounts for approximately 90% of BC-related deaths [2][3][4].
Metastatic BC, typically incurable, imposes a median survival of only 1.5 to 3 years for affected patients.
Additionally, TNBCs often develop resistance to standard-of-care treatments through unidenti ed mechanisms.Hence, preventing TNBC progression and recurrence emerges as a critical strategy to signi cantly enhance the clinical course for TNBC patients.
Given Kindlin-2's role as a coactivator of integrin activities and its interaction with TβRI, our study delves into understanding the impact of inhibiting these interactions on downstream signaling of both Integrins and TβRI, and their role in TNBC tumor progression and metastasis.Our ndings con rm direct interactions between Kindlin-2, β1-Integrin, and TβRI, with Kindlin-2 crucial for stabilizing the β1-Integrin: TβRI protein complexes.Loss of Kindlin-2 expression leads to degradation of both β1-Integrin and TβRI proteins, which can be rescued by re-expression of Kindlin-2.Importantly, loss of Kindlin-2 expression inhibits downstream signaling pathways of both β1-Integrin and TβRI.The biological signi cance of Kindlin-2-mediated stabilization is re ected in the inhibition of oncogenic behavior in TNBC tumors lacking Kindlin-2, β1-Integrin, or TβRI, both in vitro and in in vivo mouse models.
In summary, our ndings unveil a novel role for Kindlin-2 in simultaneously regulating the oncogenic activities of both β1-Integrin and TβRI by stabilizing the β1-Integrin:Kindlin-2:TβRI complex; an insight that holds promise for advancing our understanding and potential therapeutic interventions in TNBCs.

Cell lines and reagents
MDA-MB-231, 4T1, and HEK293 cells were procured from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in accordance with the manufacturer's speci ed protocols.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 to 12 months.All cells were cultured at early passages (no more than 15), and each culture was passaged no more than ve times before introducing a fresh vial.Kindlin-2, TβRI, and ITGB1-de cient cells were generated through electroporation of cancer cells with a ribonucleoprotein mixture of guide RNAs (sgRNA) and Cas9 (Synthego), following the manufacturer's instructions (Sup.Table 1).A pool of three veri ed sgRNAs was used for each human or mouse gene (Synthego), with scrambled sgRNAs serving as a negative control.

Co-Immunoprecipitation and western blotting
Cells were lysed with RIPA or NP40 lysis buffer with proteases and phosphatases inhibitor cocktails.Total protein quanti cation was performed using the BCA protein assay kit (Bio-Rad).Co-immunoprecipitation analysis involved incubating lysates at 4°C with protein A resin and speci c antibodies, as described previously [30].Western blot assays followed standard protocols with β-Actin as the loading standard.The ChemiDoc MP Imaging system (Bio-Rad) was employed for image acquisition of developed membranes.

Colony formation assay
MDA-MB-231 (3000 cells) and 4T1 (1000 cells) were seeded into 6-well plates.Cultured for 10 days, fresh medium was supplemented every 3 days.Clones were washed with PBS, xed with 4% paraformaldehyde (PFA) at room temperature for 20 minutes, and stained with 0.25% crystal violet solution.Image acquisition of the 6-well plate and quanti cation of clones were performed using the ChemiDoc MP Imaging system (Bio-Rad) and ImageJ software.

Wound healing assay
Cells were seeded in 6-well plates, grown to a con uent monolayer, and subjected to a scratch wound.After a quick wash with PBS, cells were cultured for 22 hrs.Images at 0 and 22 hrs.post-wounding were acquired using a Nikon ECLIPSE TS2r microscope, and the remaining open area was calculated using ImageJ software.

RNA Extraction and quantitative real time RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) and quanti ed with Nanodrop.Reverse transcription and quantitative real-time PCR (Bio-Rad) were performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen) and SYBR Green Master Mix Kit (Invitrogen), respectively.Primers were obtained from Qiagen (Sup.Table 2).

3D-tumorsphere and invasion assays
For 3D single-tumorsphere formation, cells were seeded into a 96-well ultralow attachment (ULA) plate and monitored for 12 days, as described previously [31].Invasion assays involved supplementing tumorspheres cultures with Matrigel, and invasion was monitored for 10 additional days.Images were captured and quanti ed using ImageJ software [31].

Cell adhesion and spreading assays
Adhesion and spreading assays were performed as described previously [31].Cells were seeded onto coverslips precoated with bronectin, laminin, and Matrigel.Adhered cells were imaged, and spreading was assessed by capturing different elds and quantifying the area around the cells using ImageJ.

Flow Cytometry analyses
Cell surface expression and activation of β1-Integrin was assessed by FACS (Sony ID7000), as described previously [31].Cells were detached using trypsin, washed, and resuspended for staining with PEconjugated mouse anti-human CD-29 antibody or FITC-conjugated HUTS-4 antibody.Data were analyzed using FlowJo software.

Immuno uorescence and confocal microscopy
Cells were seeded on glass coverslips precoated with poly-L-Lysine, processed, and incubated with primary antibodies overnight.After blocking and incubating with secondary antibodies, cells were mounted with DAPI-containing mounting medium.Images were captured on a LEICA DM5500 laser scanning confocal microscope.
In vivo tumor growth and metastasis study NSG female mice and BALB/C mice were purchased from Jackson and used for tumor growth and metastasis studies as described in our published studies [32][33][34][35].Parental and derivative cells were injected into mammary fat pads, and tumor growth was monitored.For lung metastasis assays, cells were injected into the tail vein.Mice were sacri ced, and tumors or metastases were analyzed [32][33][34][35][36].

Statistical analysis
Statistical analyses were performed using GraphPad Prism (version 8.0) and SPSS (version 21.0).All experiments were conducted in triplicate, and variables were expressed as mean ± SD.Student's t-test was used, and signi cance was considered at p < 0.05.

Kindlin-2 is overactivated in triple negative breast cancer tumors
Prior studies have established the role of Kindlin-2 as a major driver of tumor progression and metastasis in several cancers including the one that originates in the breast [15].Published studies form our group and others have shown that Kindlin-2 is involved in the activation of the oncogenic behavior of BC tumors, both in vitro and in vivo [16, 17, 20, 22-25, 27, 28, 34].Interrogation of a BC tumor microarray generated from BC specimens representing the different BC subtypes [16,37], showed high levels Kindlin-2 staining in advanced BC stages (Fig. 1A&B).Kindlin-2 staining score was signi cantly (p < 0.05) higher in BC tumors compared to normal breast tissues (Fig. 1B).More importantly, Kindlin-2 staining score was signi cantly (p < 0.05) higher in tumor of basal (hormone receptor-negative and Her2-negative) subtype (Fig. 1B), a tread that was also found in human and murine cell lines of basal and triple negative BC (TNBC) nature [16,37].These ndings were further con rmed by interrogation of public BC datasets from Oncomine (www.oncomine.org),where we found Kindlin-2 mRNA expression levels to be signi cantly (p < 0.001) higher in BC tumors compared to normal breast tissues (Fig- 1C).Furthermore, interrogation of the KM-Plotter BC database (https://kmplot.com/analysis/)showed increased expression levels of Kindlin-2 correlate with poor disease outcome in patients with BC tumors (Fig. 1D).These ndings, together with our published studies [16,17,[22][23][24]34]) support the key role that Kindlin-2 plays in the pathology of BC tumors, in general, and in TNBC tumors, in particular.One of the major functions of Kindlin-2 is the activation of the inside-out signaling of integrins through its physical interaction with the cytoplasmic tail of several integrin β-subunits, including β1-Integrin (Reviewed in [26, 38]]).Previously [16], we showed that Kindlin-2 activates the CSF1/EGF paracrine oncogenic loop in TNBC through the regulation of TGF-β signaling.Interestingly, a study by Wei et al. [29] showed that Kindlin-2 also interacts with the cytoplasmic region of TGF-β type one receptor (TβRI).Therefore, our published studies have shown that Kindlin-2 plays a major role in the regulation of TNBC tumor progression and metastasis through the regulation of the oncogenic activities of both Integrins and TGF-β.Based on this information we sought to investigate the molecular mechanisms that regulate the Kindlin-2 interaction with both β1-Integrin and TβRI, and the role of these interactions in the regulation TNBC tumor progression and metastasis.
Kindlin-2 is required for the stabilization of the β1-Integrin:Kindlin-1:TβRI protein complex Probing further into the importance of Kindlin-2 in maintaining the integrity of TβRI/Kindlin-2/β1-Integrin protein complexes, we found loss of expression of Kindlin-2 (K2-KO) in MDA-MB-231 (Fig. 2A) or 4T1 (Fig. 2B) TNBC cells leads to the degradation of both TβRI and β1-Integrin proteins.mRNA expression levels of either TβRI or β1-Integrin were not signi cantly affected in the K2-KO MDA-MB-231 (Fig. 2C) or 4T1 (Fig. 2D) TNBC cells, suggesting that loss of expression of TβRI and β1-Integrin in K2-KO cells was a result of protein degradation, but not at the mRNA transcription levels.We also used ow cytometry to measure the cell surface expression levels of β1-Integetrin and found loss of expression (KO) of either Kindlin-2, TBRI or β1-Integrin resulted in inhibition of expression of β1-Integrin expression at the surface of the cell membrane where it exerts it's signaling functions (Fig. 2E).Over-expression of full-length Kindlin-2 (K2-full) in the K2-KO MDA-MB-231 cells restored cell surface expression of β1-Integrin to levels comparable to those found in the control cells (Fig. 2F).We also used the HUTS4 assay that measures the active state of β1-Integrin [39], and found loss of expression of either Kindlin-2, TβRI or β1-Integrin resulted in inhibition of activation levels of β1-Integrin (Fig. 2G), which could also be restored by overexpressing of full-length Kindlin-2 (K2-full) in the K2-KO cells (Fig. 2H).Moreover, treating the K2-KO MDA-MB-231 (Fig. 2I) or 4T1 (2J) cells with the proteasome inhibitor MG132 resulted in the restoration of both TβRI and β1-Integrin proteins to levels found in the control cells, meanwhile over-expression of full-length Kindlin-2 in the K2-KO restored protein expression of both TβRI or β1-Integrin (Fig. KI & 2L).Therefore, these data support the role of Kindlin-2 in maintaining the physical integrity of the TβRI:Kindlin-2:β1-Integrin protein complex.
Loss of expression of either Kindlin-2, TβR1 or β1-Integrin inhibits the in vitro oncogenic behavior of TNBC tumors, and re-expression of Kindlin-2 is su cient for the restoration of these oncogenic activities To investigate the biological signi cance of loss of expression of either Kindlin-2, TβR1 or β1-Integrin (ITGB1), and their effect on the oncogenic behavior of TNBC cells, we performed several in vitro assays.Parental MDA-MB-231 or 4T1, or their Kindlin-2-, TβR1-or ITGB1-de cient (KO) derivatives were subjected to the wound healing assay (Fig. 3A-D).Loss of expression of either Kindlin-2, TβR1 or ITGB1 resulted in the inability of the MDA-MB-231 KO cells (Fig. 3A&B) or the 4T1 KO cells (Fig. 3C&D) to effectively close the scratch wound after 24 h, supporting the role of Kindlin-2, TβR1 and β1-Integrin in cancer cell migration.Loss of expression of either Kindlin-2, TβR1 or ITGB1 also inhibited the colony formation potential, a hallmark of cancer cells phenotype, of both MDA-MB-231 (Fig. 3E&F) and 4T1 (Fig. G&H) cells.To mimic the behavior of cancer cells in tumor microenvironment, we performed 3D-tumorsphere growth and tumorsphere invasion of extracellular matrices (ECMs).3D-tumorsphere growth was signi cantly (p < 0.01) inhibited in both the MDA-MB-231 (Fig. I&J and Sup Fig. 1A) and the 4T1 (3K&L and Sup. Figure 1B) cells de cient in either Kindlin-2, β1-Integrin or ITGB1 K2-KO.Similarly, MDA-MB-231 or 4T1 cells (Fig. 3M&N and Fig. 3O&P, respectively) were unable to invade ECM containing Matrigel (Fig. 3I-P, and Sup. Figure 2A&B).Interestingly re-expression of full-length Kindlin-2 (K2-Full) in K2-KO MDA-MB-231 or 4T1 cells restored their cell migration potential of MDA-MB-231 cells (Fig. 4A&B) and 4T1 cells (Fig. 4C&D).Colony formation activity was also restored in the K2-KO MDA-MB-231 and 4T1 cells reexpressing K2-Full (Fig. EG&F and Fig. 4G&H, respectively).In a similar manner, the ability of MDA-MB-231 and 4T1 cells to establish tumorspheres in 3D organoid growth assays (Fig. 4I-L, and Sup Fig. 3A&B) and for these tumorspheres to invade ECMs (Fig. 4M-P, and Sup Fig. 4A&B) were also restored in the K2-KO MDA-MB-231 and 4T1 cells re-expressing full-length Kindlin-2.These data have so far demonstrated the requirement of Kindlin-2, TβR1 and ITGB1 for the common oncogenic activities of cancer cells, and for Kindlin-2 to be su cient to restore these activities downstream of either β1-Integrin and TβRI.
Loss of expression of either Kindlin-2, TβRI or ITGB1 inhibits signaling activities that speci c to β1-

Integrin and TβRI
In the next set of experiments, we investigated the effect of loss of either Kindlin-2, TβR1 or ITGB1 on oncogenic activities that are speci cally activated downstream of either β1-Integrin or TβRI.For the β1-Integrin downstream activities, we assessed for cell adhesion on bronectin, a cellular activity that regulated by integrins [40] (Fig. 5A-H).Loss of expression of either of the three genes resulted in a signi cant (p < 0.001) inhibition of cell adhesion of MDA-MB-231 and 4T1 cells to Fibronectin (Fig. 5A&B and Fig. 5C&D, respectively).Adhesion of MDA-MB-231 and 4T1 cells to Matrigel was also signi cantly (p < 0.001) inhibited because of loss of expression of either of the three proteins (Fig. 5E&F and Fig. 5G&H, respectively).Cell adhesion to Laminin was also affected as a result of loss of expression of either of three genes (Sup. Figure 5).Cell spreading on ECM is also a cellular activity that is tightly regulated by integrins [40].Here again, we found loss of expression of either Kindlin-2, TβR1 or ITGB1 resulted in a signi cant (p < 0.001) inhibition of spreading of MDA-MB-231 and 4T1 to bronectin (Fig. 5I&J and Fig. 5K&L, respectively) to Matrigel (Fig. 5M&N and Fig. O&P, respectively), or to Laminin (Sup. Figure 6).Thus we show loss of expression of either Kindlin-2, TβR1 or ITGB1 to inhibits cellular activities that are speci c to Integrins.Since β1-Integrin can form heterodimers with other α-Integrin subunits [41][42], we used qt-RT-PC to assess for expression levels of α1, αV and α5, which are predominantly expressed in cancer cells [43].Loss of Kindlin-2 did not affect expression levels of either α1, αV and α5 subunits (Sup.Figure 7A).However, loss of either ITGB1 or TβR1 resulted in a signi cant (p < 0.01) inhibition of expression of both αV and α5 subunits, but those of α1 subunit (Sup.Figure 7B and 7C, respectively).This is consistent with the spreading data obtained on Fibronectin and Lamin since both αV and α5 integrin subunits, but not α1 subunit are involved in the β1-Integrin-mediated interaction with Fibronectin and Laminin [42].Finally, as a readout for the molecular signaling downstream of TβRI, we assessed for phosphorylation levels of SMAD2/3 that takes place downstream of the TGF-β-mediated activation of the TβRI:TβRII complex.Loss of expression of either of the three proteins also resulted in reduction in phosphorylation levels of SMAD2/3 in both MDA-MB-231 (Fig. 5Q) and 4T1 cells (Fig. 5R).Levels of basal pSMAD2/3 did not increase in the KO cells even after stimulation of TGF-β (Sup. Figure 8A).Phosphorylation levels of TβRII, the upstream effector of SMAD, were also inhibited in the KO cells (Sup. Figure 8B).Thus, we show that loss of expression of either Kindlin-2, TβR1 or ITGB1 inhibited the TβRI-speci c downstream signaling, and, therefore, implicating Kindlin-2 as a major player in the regulation of the downstream signaling effectors of both β1-Integrin and TβR1.
Re-expression of Kindlin-2 in the K2-de cient TNBC cells is su cient for the restoration of the oncogenic activities downstream of β1-Integrin and TBRI To determine whether the cellular activities that are regulated downstream of β1-Integrin are mediated by Kindlin-2, the K2-KO MDA-MB-231 and 4T1 TNBC cells re-expressing full-length Kindlin-2 were subjected to cell adhesion and spreading.Cell adhesion on bronectin was fully restored in both K2-de cient MDA-MB-231 cells (Fig. 6A&B) and 4T1 cells (Fig. 6C&D) re-expressing Kindlin-2.Cell adhesion on Matrigel was also fully restored for both cell lines (Fig. 6E&F, and Fig. 6G&H), as well as Laminin (Sup. Figure 9).Similarly, re-expression of full length Kindlin-2 in the K2-KO cells also fully restored the spreading potential of both cell lines on both bronectin (Fig. 6I&J and Fig. 6K&L for MDA-MB-231 and 4T1, respectively), Matrigel (Fig. 6M&N and Fig. 6O&P for MDA-MB-231 and 4T1), and Laminin (Sup. Figure 10).Phosphorylation levels of SMAD, a readout of TβRI-speci c downstream signaling activity, was also restored in K2-KO MDA-MB-231 cells treated with the proteosome inhibitor MG132 (Fig. 6Q), as well as in the K2-KO cells re-expressing full length Kindlin-2 (Fig. 6R).Thus we con rm that the signaling activities that are mediated downstream of β1-Integrin or TBRI are speci cally regulated by Kindlin-2.additionally, we con rmed that Kindlin-2 expression, by stabilizing the β1-Integrin:TβRI protein complex is su cient for the restoration of these β1-Integrin and TβRI downstream cellular and signaling activities.
Loss of expression of either Kindlin-2, TβRI or ITGB1 inhibits growth and metastasis of TNBC tumors, which can be restored buy re-expression of Kindlin-2 Next, we determined whether the biological and signaling effects observed in vitro, can also be recapitulated in vivo in mouse models for TNBC tumor progression and metastasis.Using the spontaneous metastasis mouse model, MDA-MB-231 or 4T1 TNBC control cells and their KO derivatives were injected in the mammary fat pads of NSG mice (MDA-MB-231) or Balb/C mice (4T1), and growth of the primary tumors was monitored over time.Loss of expression of either of the three proteins resulted in a signi cant (p < 0.001) delay in tumor growth and weight in both the MDA-MB-231 model (Fig. 7A&B) as well as the 4T1 model (Fig. 7C&D).Metastasis was also signi cantly (p < 0.001) inhibited in the lungs of mice injected with the KO MDA-MB-231 cells (Fig. 7E&F) or with KO 4T1 cells (Fig. G&H).Re-expression of Kindlin-2 in the Kindlin-2-de cient MDA-MB-231 cells resulted in the rescue of both tumor growth (Fig. 7I) and metastasis (Fig. 7J&K).These data show the impact of Kindlin-2, TβRI and ITGB1on tumor progression and metastasis.They also con rm the speci city of Kindlin-2 in the process, where Kindlin-2 is su cient for the restoration of the growth and metastasis potentials of TNBC tumors that lack expression of either TβRI or ITGB1.

Discussion
Kindlin-2 initially garnered attention for its pivotal role in activating integrins, thereby mediating cellextracellular matrix adhesion and signaling [24,[44][45][46][47][48][49].This function is crucial for facilitating the interaction between cells and their extracellular environment by modulating integrin activity [24,[44][45][46][47][48][49].Integrins, as transmembrane receptors, play a key role in cell adhesion and signal transduction.Kindlin-2 binds to the cytoplasmic tails of integrins, promoting their activation and enabling interaction with extracellular ligands.This activation is vital for cell adhesion, migration, and communication with the surrounding microenvironment.The dynamic interplay between Kindlin-2 and integrins signi cantly contributes to cell behavior under both normal physiological conditions and pathological manifestations such as cancer.In normal cellular functions, this relationship is crucial for embryonic development, tissue homeostasis, and immune responses [15].However, dysregulation of Kindlin-2 and integrin interactions has been linked to various diseases, particularly cancer, where aberrant cell adhesion and migration are prominent features [13].
Beyond its role in integrin activation, Kindlin-2 has emerged as a key player in regulating transforming growth factor-beta (TGF-β) signaling, the activating ligand of the TβRI:TβRII signaling complex [29,50].This interplay between Kindlin-2 and TGF-β regulates various cellular processes and contributes to both normal development and pathological conditions, including breast cancer (BC) pathology [16].The Kindlin-2-TGF-β axis plays a crucial role in epithelial-mesenchymal transition (EMT), a process central to embryonic development and implicated in cancer metastasis [17,51].Dysregulation of TGF-β signaling is a hallmark of cancer progression, and Kindlin-2 has been implicated in mediating TGF-β effects on tumor cell behavior [16,17].Notably, the interaction between Kindlin-2 and TGF-β receptors enhances cellular responsiveness to TGF-β and its downstream signaling, in uencing processes such as cell proliferation and differentiation [29,52].In turn, TGF-β signaling also regulates Kindlin-2 expression.
Previously, our studies demonstrated that Kindlin-2 activates the CSF1/EGF paracrine oncogenic loop in BC through the regulation of TGF-β signaling [16].Additionally, a study by Wei et al. [29] revealed Kindlin-2's binding to the cytoplasmic region of the TGF-β type one receptor (TβRI).Consequently, Kindlin-2 plays a major role in regulating triple negative breast cancer (TNBC) tumor progression and metastasis through the modulation of the oncogenic activities of both integrins and TGF-β.
In this paper, we present, for the rst time to the best of our knowledge, the direct binding of Kindlin-2 to TβRI and β1-Integrin, establishing a physical bridge between TβRI and β1-Integrin.Moreover, Kindlin-2 not only is necessary for the stabilization of the TβRI:Kindlin-2:β1-Integrin complex but is also required to maintain the oncogenic behavior of TNBC cell lines both in vitro and in vivo.Indeed, Kindlin-2 is a crucial protein involved in integrin-mediated adhesion and signaling processes, essential for cell-extracellular matrix adhesion [53].Kindlin-2 is believed to associate with β1-Integrin at nascent adhesions before talin recruitment during adhesion maturation, indicating its early involvement in the adhesion process [54] (Sup.Figures 11 and 12).Disrupting the interaction between the Kindlin-2 dimer inhibits adhesion formation, integrin activation, and cell spreading, underscoring the signi cance of Kindlin-2 in these processes [38,[55][56][57].
Conversely, TβRI plays a vital role in the TGF-β/SMAD signaling pathway, regulating cell growth, differentiation, and migration, making it a central mediator of cancer progression [58].Studies on TβR1 and SMAD in breast cancer emphasize the multifaceted role of the TGF-β signaling pathway and its components in the disease.While genetic variants such as TβRI*6A have shown associations with breast cancer risk and progression, the regulatory mechanisms and functional implications of TβR1 and SMAD signaling in breast cancer require further investigation for a comprehensive understanding of their potential as therapeutic targets.Our study demonstrates that the loss of expression of Kindlin-2, TβR1, or β1-Integrin proteins results in a reduction in phosphorylation levels of SMAD2/3 in both MDA-MB-231 and 4T1 cells.Activated TβR1 phosphorylates SMAD2 and SMAD3, which heterodimerize with SMAD4 and translocate to the nucleus, binding to DNA and regulating the transcription of target genes involved in various cellular functions [59].A recent study has also reported on the interplay between β1-Integrin, Kindlin-2, and TBRI partner, TBRII, to promote pancreatic tumor growth [52].This study establishes the molecular mechanism by which the interplay between these oncogenic proteins is regulated in the context of breast cancer progression and metastasis.
Our investigation employed a combination of in vitro assays, two different mouse models for TNBC tumors, as well as genetic and pharmacological manipulation to establish Kindlin-2's role as a major contributor to the stabilization of the TβRI:β1-Integrin protein complexes and the regulation of their downstream oncogenic activities, driving the progression and metastasis of TNBC tumors.We show that Kindlin-2 establishes direct interactions with TβRI and β1-Integrin, with the interaction between Kindlin-2 and TβRI mediated through the F2 domain of Kindlin-2 and the interaction between Kindlin-2 and β1-Integrin mediated through the F3 domain of Kindlin-2 via a QW amino acid doublet.CRISPR/Cas9mediated knockout of Kindlin-2 leads to the loss of both TβRI and β1-Integrin proteins, resulting in the destabilization of the protein complex, which can be rescued by inhibiting the proteasome degradation machinery or re-expressing full-length Kindlin-2 in Kindlin-2-de cient cells.This supports the novel function of Kindlin-2 in establishing a physical bridge between TβRI and β1-Integrin and the requirement of Kindlin-2 for the stabilization of this complex.Loss of expression of either Kindlin-2, TβRI, or β1-Integrin leads to the inhibition of in vitro and in vivo oncogenic behavior of TNBC cells, highlighting the importance of either member of this protein complex in maintaining the oncogenic behavior of cancer cells; loss of one member of this complex is su cient for the mitigation of the oncogenic activities of cancer cells.The downstream signaling effectors speci c to either TβRI (phosphorylation of SMAD2/3) or β1-Integrin (cell adhesion and spreading on bronectin) can be inhibited by simply losing Kindlin-2, emphasizing the speci city of Kindlin-2 in modulating TβRI-and β1-Integrin-mediated regulation of the oncogenic behavior of cancer cells.
Our study unveils, for the rst time, the intricate relationship between Kindlin-2, integrins, and TβRI, regulating crucial cellular processes (Fig. 8).This trilateral interplay integrates the roles of focal adhesion protein Kindlin-2, transmembrane receptors integrins, and TβRI in orchestrating various physiological and pathological events.At the core of this relationship lies the modulation of integrin activation by Kindlin-2.Kindlin-2 interacts with the cytoplasmic tails of integrins, promoting their activation and facilitating the connection between cells and the extracellular matrix (ECM).This activation is essential for cellular adhesion and sets the stage for downstream signaling events.On the other hand, Kindlin-2 in uences the TGF-β signaling pathway through its interaction with TβRI, enhancing its activation and promoting downstream TGF-β signaling cascades.This interaction contributes to the regulation of cellular processes such as proliferation, differentiation, and migration, emphasizing the central role of Kindlin-2 in integrating signals from both integrins and TGF-β.Importantly, in a clinical setting, dysregulation of this Kindlin-2:Integrins:TβRI axis has implications in diseases such as cancer, where aberrant integrin activation and TGF-β signaling are hallmarks of cancer progression, and Kindlin-2 emerges as a potential key player bridging these pathways.

Conclusions
In conclusion, the β1-integrin:Kindlin-2:TβRI interconnection represents a sophisticated network that regulates BC progression and metastasis.Further investigations of this trilateral oncogenic axis holds promise for deciphering the intricacies of cellular behavior in disease, and therefore offering potential therapeutic avenues to speci cally target these pathways for the treatment of TNBC tumors and other tumors.
Abbreviations BC: Breast cancer

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