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 significantly (p < 0.05) higher in BC tumors compared to normal breast tissues (Fig. 1B). More importantly, Kindlin-2 staining score was significantly (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 findings were further confirmed by interrogation of public BC datasets from Oncomine (www.oncomine.org), where we found Kindlin-2 mRNA expression levels to be significantly (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 findings, together with our published studies [16, 17, 22–24, 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 significantly 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 flow 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 over-expressing 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 sufficient for the restoration of these oncogenic activities
To investigate the biological significance 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-deficient (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 significantly (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 deficient 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 re-expressing 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 sufficient 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 specific 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 specifically activated downstream of either β1-Integrin or TβRI. For the β1-Integrin downstream activities, we assessed for cell adhesion on fibronectin, a cellular activity that regulated by integrins [40] (Fig. 5A-H). Loss of expression of either of the three genes resulted in a significant (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 significantly (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 significant (p < 0.001) inhibition of spreading of MDA-MB-231 and 4T1 to fibronectin (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 specific 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 significant (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-specific 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-deficient TNBC cells is sufficient 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 fibronectin was fully restored in both K2-deficient 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 fibronectin (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-specific 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 confirm that the signaling activities that are mediated downstream of β1-Integrin or TBRI are specifically regulated by Kindlin-2. additionally, we confirmed that Kindlin-2 expression, by stabilizing the β1-Integrin:TβRI protein complex is sufficient 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 significant (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 significantly (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-deficient 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 confirm the specificity of Kindlin-2 in the process, where Kindlin-2 is sufficient for the restoration of the growth and metastasis potentials of TNBC tumors that lack expression of either TβRI or ITGB1.