Silencing LAMC2 promotes ER stress, apoptosis, and mitochondrial dysfunction

The oncogenic capacity of LAMC2 has been well-documented in lung cancer, undifferentiated thyroid carcinoma, cholangiocarcinoma, laryngeal cancer, ovarian cancer and pancreatic cancer. We began our study by examining the correlation between LAMC2 and pan-cancer using The Cancer Genome Atlas (TCGA) database. As our results indicated, of 33 different cancer types, LAMC2 is overexpressed in 21, reduced in 7, and unassociated with five cancer types (Supp. Fig. 1A). In addition, we examined the correlation between LAMC2 and overall survival for each cancer type. Results showed that in LAMC2 overexpressing tumors, LAMC2 is negatively correlated with overall survival (Supp. Fig. 1B). In addition, Progression Free Interval (PFI, Supp. Fig. 1C) and Disease Specific Survival (DSS, Supp. Fig. 1D) analyses showed similar trends. These results suggested that LAMC2 is positively associated with tumor progression and poor prognosis.

In addition, pan-cancer Gene Set Enrichment Analysis (GSEA) analysis was also carried out using the TCGA database for the same 33 cancer types. LAMC2 was found significantly correlated with unfolded protein response, which is a crucial ER stress adaptive response mechanism, as well as physiological processes including reactive oxygen species (ROS) generation and oxidative phosphorylation (Fig. 1A left). ROS production and oxidative phosphorylation are important biological processes related to the mitochondria. Therefore, we performed another GSEA analysis to examine the potential correlation between LAMC2 and mitochondria-specific signaling pathways. Results indicated that LAMC2 was negatively correlated with oxidative phosphorylation pathways, and positively correlated with mitochondrial dynamics and surveillance, as well as apoptosis (Fig. 1A, right). These data indicated that LAMC2 is involved in ER stress- and mitochondrial-related biological processes. To verify the function of LAMC2 in ER stress response, we first confirmed subcellular localization of LAMC2 in the ER using live-cell fluorescence detection. As shown in Fig. 1B, LAMC2 can be found to localize in the ER of A549, MCF7, and MHCC-97H cells. In addition, tunicamycin (Tun) and Bredfeldin A (BFA) treatment, which were used to induce ER stress, significantly promoted LAMC2 and GRP78 (ER stress marker) protein levels (Fig. 1C). Our data also showed that GRP78 and LAMC2 protein levels increased with increasing treatment time (Supp. Fig. 2D). Moreover, mRNA levels of LAMC2 were also increased post tunicamycin treatment (Supp. Fig. 2B). To explore whether LAMC2 is involved in ER stress-induced ROS production, mitochondrial dysfunction, and consequent apoptosis, we inhibited LAMC2 expression in A549, MCF7, and MHCC-97H cells using siRNA. Flow cytometry results indicated that LAMC2 silencing significantly induced early and late apoptosis (Fig. 1D), as well as ROS production (Fig. 1E). Moreover, while the proportion of JCI polymer cells decreased, monomer cells increased in si-LAMC2 cells, suggesting that silencing LAMC2 led to a reduction in mitochondrial membrane potential (Fig. 1F). Live-cell fluorescence staining further confirmed these results (Fig. 1G and Supp. Fig. 3A–C). Additionally, western blotting analyses showed that LAMC2 knockdown led to increased protein levels of Cleaved-PARP, Cleaved-caspase9, Cleaved-caspase6, and BAX, which are known to promote apoptosis. On the other hand, BCL2 protein levels were reduced. In addition, ER stress proteins GRP78, ATF6, PERK, and phosphorylated PERK were upregulated in LAMC2 knockdown cells. Interestingly, IRE1 protein level was unaffected (Fig. 1H). The above results demonstrated that ER stress induced abnormal elevation of LAMC2 protein levels. Repression of LAMC2, however, elicited ER stress and impaired mitochondrial function, ultimately triggering apoptosis in cancer cells. Collectively, our data suggested that under ER stress, LAMC2 protein level is upregulated to enhance mitochondrial function and mitigate stress-induced cell death.

Fig. 1: Silencing LAMC2 promotes ER stress, apoptosis, and mitochondrial dysfunction.

A TCGA pan-cancer database was used to analyze hallmarks (left) and mitochondria-related pathways (right) significantly correlated with LAMC2 expression. B Live-cell fluorescence images of co-localization between LAMC2 (green) and ER (blue) in A549, MCF7, and MHCC-97H cells. C Western blots of LAMC2, GRP78, and GAPDH in A549, MCF7, and MHCC-97H cells treated with Tun or BFA. D Annexin V/FITC flow cytometry analysis of early and late cellular apoptosis in LAMC2 knockdown cancer cells. E Flow cytometry analysis of ROS production in LAMC2 knockdown cancer cells. F Flow cytometry analysis of mitochondrial membrane potential in LAMC2 knockdown cells represented by polymer and monomer levels. G Live-cell fluorescence imaging was used to detect Annexin V/FITC, polymer JC1, monomer JC1, and ROS in A549, MCF7, and MHCC-97H cells. H Western blots of PARP, cleaved-PARP, caspase 9, cleaved caspase 9, caspase 6, cleaved caspase 6, Bax, BCL2, IRE1, p-PERK, PERK, ATF6, GRP78, LAMC2, and GAPDH protein levels. Graphical data were presented as mean± SEM. *p < 0.05, **p < 0.01.

LAMC2 mitigates ER stress and is associated with ER-mitochondria interaction

To confirm the role of LAMC2 in ER stress response, we treated LAMC2 overexpressing A549 and MCF7 cells with tunicamycin and evaluated the effect of ER stress on apoptosis, ROS production, and mitochondrial membrane potential. As shown in Fig. 2A, Tun-induced apoptosis, ROS production, and reduction in membrane potential were significantly lower in the LAMC2 overexpressing cells compared to the control cells. Moreover, BFA- and Tun-induced increase in ER stress makers such as PERK, ATF6, CHOP, and GRP78 were also significantly less in the LAMC2 overexpressing group (Fig. 2B). These results suggested that LAMC2 overexpression can reverse ER stress-induced cell apoptosis, as well as increase ROS production and decrease mitochondria membrane potential. Furthermore, treatment of A549 cells with si-LAMC2 significantly enhanced the induction of endoplasmic reticulum stress markers (GRP78, PERK, and ATF6) by Tun compared to the control group. Consistent with previous findings, LAMC2 knockdown resulted in increased cell apoptosis under non-Tun treatment conditions when compared to the control group. Moreover, under Tun treatment conditions, the extent of cell apoptosis was further augmented in the LAMC2 knockdown group relative to the control group (Supp. Fig. 4C, D). Having validated the impact of LAMC2 on both ER stress and mitochondria-related biological processes, we next examined potential changes in the physiological interaction between ER and mitochondria under ER stress using live-cell fluorescent staining. Results indicated that compared to the control group, Tun treated cells showed significantly higher ER-mitochondria interaction (Fig. 2C, D). To corroborate these results, we showed that Tun treatment also promoted dynamin-related protein 1 (DRP1) deposition near the nucleus (Fig. 2E). DRP1 is an important player in regulating mitochondria dynamics, and has been shown to facilitate ER and mitochondria interaction [21]. Therefore, our results validated that Tun-induced ER stress promoted ER-mitochondria interaction at the perinuclear region. Noted, increase in ER and mitochondria interaction has been previously reported as a potential mechanism for relieving ER stress [22,23,24,25].

Fig. 2: LAMC2 mitigates ER stress and is associated with ER-mitochondria interaction.

A Flow cytometry analyses of Annexin V/FITC, ROS, and JC1-FITC in control and LAMC2 overexpressing A549 and MCF7 cells treated with Tun. B Western blots of PERK, ATF6, CHOP, GRP78, LAMC2-GFP, and GAPDH protein levels in control and LAMC2 overexpressing A549 and MCF7 cells treated with either BFA or Tun. C Live-cell fluorescence images of co-localization between ER (green) and mitochondria (red) in A549, MCF7, and MHCC-97H cells treated with DMSO or Tun. D Quantification of ER-mitochondria co-localization. E Immunofluorescence images of DRP1(red) and DAPI (blue) in A549, MCF7, and MHCC-97H cells. F Live-cell fluorescence images of LAMC2-GFP (green), mitochondria (red), and ER (blue), as well as co-localization between LAMC2/mitochondria, LAMC2/ER, and LAMC2/mitochondria/ER in A549, MCF7, and MHCC-97H cells. GFP was used as control. Graphical data were presented as mean ± SEM. *p < 0.05, **p < 0.01.

In effort to determine whether LAMC2 participates in enhancing ER-mitochondria interaction, we transfected GFP-tagged LAMC2 vectors into A549, MCF7, and MHCC-97H cells, and examined the co-localization between LAMC2, ER, and mitochondria using live-cell fluorescence microscopy. Imaging results showed that LAMC2 co-localized strongly with ER and mitochondria at the ER-mitochondria junction, which is also where DRP1 can be found (Fig. 2F). In contrast, GFP in the control group localized in a diffused and non-specific manner. Overall, our data demonstrated that LAMC2 not only participates in preventing ER stress-induced apoptosis and mitochondria dysfunction, but can also physically interact with ER and mitochondria at the ER-mitochondria junction. These results suggested that LAMC2 may mitigate ER stress at least in part via promoting ER-mitochondria interaction.

LAMC2 interacts with MYH9 and MYH10

To further explore the underlying mechanism of LAMC2 in regulating ER- and mitochondria-related biological functions, we first examined the potential binding partners of LAMC2 in A549 cells through immunoprecipitation coupled to mass spectrometry (IP-MS). As shown in Fig. 3A, B, most of the proteins bound by LAMC2 are cytoskeletal proteins. Silver staining results in MCF7 and MHCC-97H cells are shown in Supplementary Fig. 4E. KEGG pathway analysis showed that the main biological processes associated with the top 20 proteins with highest binding affinity to LAMC2 include localization, response to stimulus, and organelle interaction (Fig. 3C). Likewise, GO and protein-protein interaction (PPI) analyses revealed that LAMC2-bound proteins are highly associated with ER processing and multi-organism metabolic processes (Supp. Fig. 4F–H). Collectively, these results confirmed that LAMC2 and its binding partners are highly correlated with ER-related biological processes. Among the top binding proteins, we found MYH9 and MYH10 proteins to be tightly associated with ER and mitochondria dynamics [26]. Thus, we examined the physical interaction between MYH9, MYH10, and LAMC2 through immunofluorescence imaging. In both A549 and MCF7 cells, LAMC2 was found to co-localize with both MYH9 and MYH10 near the nuclear membrane (Fig. 3D). In addition, co-immunoprecipitation (Co-IP) experiments confirmed the ability for LAMC2 to bind both MYH9 and MYH10 (Fig. 3E). Interestingly, in addition to separately interacting with LAMC2, Co-IP results also demonstrated the physical interaction between MYH9 and MYH10 (Fig. 3F). These results suggested that LAMC2, MYH9, and MYH10 proteins can exist in the form of a complex. To further validate the binding relationship between LAMC2, MYH9, and MYH10, we transfected His-tagged LAMC2 plasmid into A549 and MCF7 cells. Co-IP results confirmed the binding interaction between MYH9, MYH10, and exogenous LAMC2 (Fig. 3G). As previously mentioned, MYH9 and MYH10 are important for ER and mitochondrial dynamics. Therefore, binding between LAMC2, MYH9, and MYH10 may be involved in regulating ER-mitochondria interaction.

Fig. 3: LAMC2 interacts with MYH9 and MYH10.

A Silver staining image of proteins co-purified with LAMC2. B Table showing top proteins with high affinity for LAMC2. C KEGG pathway analysis of biological processes associated with top 20 proteins with highest binding affinity to LAMC2. D Immunofluorescence images of DAPI (blue), LAMC2 (green), and MYH9/MYH10 (red), as well as co-localization between LAMC2 and MYH9, LAMC2 and MYH10 in A549 and MCF7 cells. E Co-immunoprecipitation of LAMC2 with MYH9 and MYH10 in A549 and MCF7 cells. F Co-immunoprecipitation of MYH9 and MYH10 with LAMC2 in A549 and MCF7 cells. G Co-immunoprecipitation of LAMC2-His with MYH9 and MYH10 in A549 and MCF7 cells.

ER stress stimulates the formation of LAMC2/MYH9/MYH10 protein complex

To examine potential changes in MYH9 and MYH10 under ER stress, we treated A549, MCF7, and MHCC-97H cells with various concentrations of tunicamycin, and found that protein levels of MYH9, MYH10, LAMC2, GRP78, as well as DRP1 significantly increased (Fig. 4A, B). In addition, qRT-PCR results showed that MYH9 and MYH10 mRNA levels were also slightly increased (Supp. Fig. 2B). Considering that GAPDH is associated with metabolism, its use is suitable for quantitative analysis of cytoskeletal proteins. However, since DRP1 is a key protein associated with mitochondria metabolism, we chose to repeat the western blotting analyses using β-actin as the internal reference (Fig. 4B). As mentioned earlier, ER-mitochondria localization at the perinuclear region is an indication of ER stress response. Corroborating this phenomenon, our immunofluorescence imaging results indicated that tunicamycin treatment significantly enhanced the co-localization between LAMC2, MYH9, and MYH10 near the nuclear membrane (Fig. 4C). Quantification of the fluorescent images confirmed this finding. In addition, Co-IP experiments showed that the increased binding of MYH9 to LAMC2 and MYH10 after tunicamycin treatment was more evident in the LAMC2 overexpressing A549 cells compared to the control cells (Fig. 4D, left). Similarly, binding of MYH10 to LAMC2 and MYH9 after tunicamycin treatment was more significant in the LAMC2 overexpressing cells (Fig. 4D, right). Consistent results were also found in MCF7 cells (Fig. 4E). These results suggested that ER stress enhanced protein binding between LAMC2, MYH9, and MYH10. To examine the effect of LAMC2 on MYH9 and MYH10, we performed western blotting analyses using si-LAMC2 cancer cells, and showed that silencing LAMC2 significantly reduced the protein levels of MYH9, MYH10, as well as DRP1 and phosphorylated DRP1 (Fig. 4F). However, qRT-PCR results demonstrated that mRNA levels of MYH9 and MYH10 were not changed in si-LAMC2 cells (Supp. Fig. 2A), which suggests that the interaction between LAMC2, MYH9, and MYH10 is mainly at the protein level. Collectively, our data demonstrated that ER stress enhances LAMC2 expression and promotes LAMC2/MYH9/MYH10 protein complex formation near the nuclear membrane, which may be the underlying mechanism for facilitating ER-mitochondria interaction and alleviating ER stress-induced apoptosis.

Fig. 4: ER stress stimulates the formation of LAMC2/MYH9/MYH10 protein complex.

A Western blots of DRP1, MYH10, MYH9, LAMC2, and GRP78 with GAPDH as internal reference for A549, MCF7, and MHCC-97H cells treated with Tun. B Western blots of DRP1, MYH10, MYH9, LAMC2, and GRP78 with β-actin as internal reference for A549, MCF7, and MHCC-97H cells treated with Tun. C Immunofluorescence images of DAPI (blue), LAMC2 (green), MYH9/MYH10 (red), as well as co-localization between LAMC2 and MYH9, LAMC2 and MYH10 in A549, MCF7, and MHCC-97H cells treated with either DMSO or Tun. Quantification graphs demonstrate co-localization between LAMC2/MYH9 and LAMC2/MYH10 (bottom). D Co-immunoprecipitation of LAMC2 with MYH9 and MYH10 in control and LAMC2 overexpressing A549 cells treated with Tun. E Co-immunoprecipitation of LAMC2 with MYH9 and MYH10 in control and LAMC2 overexpressing MCF7 cells treated with Tun. F Western blots of P-DRP1, DRP1, MYH10, MYH9, LAMC2, and GAPDH in control and LAMC2 knockdown cancer cells (left). Western blots of P-DRP1, DRP1, MYH10, MYH9, LAMC2, and β-actin protein levels in control and LAMC2 knockdown cancer cells (right). Graphical data were presented as mean± SEM. *p < 0.05, **p < 0.01.

LAMC2 overexpression promotes ER-mitochondria interaction

As previously shown in Fig. 2C, tunicamycin treatment promoted ER-mitochondria co-localization. To establish that this phenomenon is driven by LAMC2 overexpression, we used live-cell fluorescent imaging to examine ER-mitochondria co-localization in LAMC2 silencing cells. As shown in Fig. 5A, while tunicamycin treatment enhanced ER-mitochondria co-localization, the degree of co-localization was significantly lower in the si-LAMC2 cells. This result suggested that LAMC2 expression is important for promoting ER-mitochondria interaction at the perinuclear region. In addition, we showed that LAMC2 overexpression and tunicamycin treatment synergistically promoted protein levels of DRP1, MYH9, and MYH10 in A549 and MCF7 cells (Fig. 5B). Using RHOD2-AM fluorescent staining, we also showed that silencing LAMC2, MYH9 or MYH10 inhibited mitochondria calcium levels (Fig. 5C, D). Furthermore, we detected the degree of co-localization between LAMC2, mitochondria, and ER under tunicamycin and Mdivi-1 treatment. As a mitochondrial-division inhibitor, Mdivi-1 can be used to inhibit DRP1 levels, which would subsequently hinder ER-mitochondria interaction [21, 27]. As shown in Fig. 5E, while tunicamycin treatment promoted LAMC2 co-localization with the mitochondria and ER, Mdivi-1 reduced binding between the three. Moreover, while Mdivi-1 treatment reduced the protein levels of MYH9, MYH10, P-DRP1, and DRP1, overexpression of LAMC2 reversed this effect (Fig. 5F). Lastly, we treated control and LAMC2 overexpressing cells with either Mdivi-1 or Mdivi-1+Tun to examine potential changes in cell apoptosis via flow cytometry. As demonstrated in Fig. 5G, while Mdivi-1 alone can promote early and late apoptosis, the effect was significantly more evident in the combined treatment (MDIVI-1+Tun) group. Compared to the control group, LAMC2 overexpression was able to reverse Mdivi-1 and Tun-induced cell apoptosis. Overall, our data suggested that upon ER stress, LAMC2 expression is elevated in part to promote ER-mitochondria interaction.

Fig. 5: LAMC2 overexpresison promotes ER-mitochondria interaction.

A Live-cell fluorescence images of co-localization between ER (green) and mitochondria (red) in control and LAMC2 knockdown cancer cells treated with either DMSO or Tun. Quantification of ER-mitochondria co-localization (right). B Western blots of DPR1, MYH10, MYH9, LAMC2, GAPDH, and β-actin protein levels in control and LAMC2 overexpressing A549 and MCF7 cells treated with Tun. C Mitochondrial calcium levels detected by Rhod-2-AM Cell Permeable Kit in control and LAMC2 knockdown cancer cells. D Graphical representation of Rhod-2-AM fluorescence A.U.C in control and LAMC2 knockdown cancer cells. E Live-cell fluorescence images of LAMC2 (green), mitochondria (red), and ER (blue), as well as co-localization between LAMC2/mitochondria, LAMC2/ER, and LAMC2/mitochondria/ER in A549, MCF7, and MHCC-97H cells treated with DMSO, Tun, or MDIVI-1. Quantification of LAMC2/Mitochondria and LAMC2/ER co-localization (right). F Western blots of MYH10, MYH9, P-DRP1, DRP1, LAMC2, and β-actin protein levels in control and LAMC2 overexpressing cancer cells treated with MDIVI-1. G Flow cytometry analysis of early and late cellular apoptosis in control and LAMC2 overexpressing cancer cells treated with DMSO, MDIVI-1, or Mdivi-1+Tun. Graphical data were presented as mean± SEM. *p < 0.05, **p < 0.01.

MYH9 and MYH10 regulates ER stress and mitochondrial function

While previous studies have indicated that MYH9 and MYH10 are important for mitochondria dynamics, and may play a role in ER stress, it is unclear whether they participate in similar cellular physiological processes as LAMC2, including ROS production and mitochondrial membrane potential maintenance. Through flow cytometry analyses, we measured ROS production and mitochondrial membrane potential in MYH9 and MYH10 knockdown cells. As shown in Fig. 6A, B, silencing MYH9 and MYH10 significantly promoted ROS production, and reduced mitochondrial membrane potential. In addition, we showed that MYH9 and MYH10 knockdown cancer cells exhibited lower colony formation and cell viability compared to the control group cells (Fig. 6C, D). Live-cell fluorescent imaging confirmed the increase in ROS production and decrease in mitochondrial membrane potential in MYH9 and MYH10 knockdown cells (Fig. 6E, F and Supp. Fig. 3D, E). Lastly, through western blotting analyses, we showed that silencing MYH9 and MYH10 promoted ER stress markers such as GRP78, ATF6, PERK, and P-PERK (Fig. 6G). On the other hand, BCL2 was reduced while BAX was elevated in MYH9 and MYH10 knockdown cells, indicating reduced cell apoptosis. In addition, knockdown of MYH9 promoted LAMC2 but inhibited MYH10 protein levels. Similarly, knockdown of MYH10 promoted LAMC2 but reduced MYH9 protein levels. The increase in LAMC2 protein may be a result of si-MYH9- or si-MYH10-induced ER stress. At the mRNA level, knockdown of MYH9 in A549 and MHCC-97H cells promoted LAMC2 mRNA levels but MYH10 mRNA levels remained unchanged. Similarly, knockdown of MYH10 elevated LAMC2 mRNA levels, while MYH9 mRNA levels remained unchanged (Supp. Fig. 2C). However, in MCF7 cells, knockdown of MYH9 promoted the mRNA levels of both LAMC2 and MYH10. Same were found for MYH10 knockdown cells (Supp. Fig. 2C). The varied qRT-PCR results further suggested that the interaction between LAMC2, MYH9, and MYH10 are mainly at the protein level. Overall, data from this section confirmed that LAMC2, MYH9, and MYH10 exist in a complex, and that knockdown of any one of the proteins will affect the protein levels of the other two.

Fig. 6: MYH9 and MYH10 regulates ER stress and mitochondrial function.

A Flow cytometry analysis of ROS production in MYH9 and MYH10 knockdown cancer cells. B Flow cytometry analysis of mitochondrial membrane potential in MYH9 and MYH10 knockdown cells represented by polymer and monomer levels. C Colony formation of MYH9 and MYH10 knockdown cancer cells. Quantification of clone number (bottom). D Cell activity levels of MYH9 and MYH10 knockdown cancer cells. E Live-cell fluorescence imaging was used to ROS in MYH9 and MYH10 knockdown cancer cells. F Live-cell fluorescence imaging was used to polymer JC1 and monomer JC1 in MYH9 and MYH10 knockdown cancer cells. G Western blots of LAMC2, P-PERK, PERK, BCL2, BAX, ATF6, GRP78, MYH10, MYH9, and GAPDH in MYH9 and MYH10 knockdown cancer cells. Graphical data were presented as mean ± SEM. *p < 0.05, **p < 0.01.

LAMC2 enhances ER-mitochondria interaction and mitigates ER stress through binding to MYH9 and MYH10

To establish the effect of MYH9 and MYH10 on ER-mitochondria interaction, we knocked down either MYH9 or MYH10 in A549, MCF7, and MHCC-97H cells. Using live-cell fluorescence imaging, we showed that silencing either MYH9 or MYH10 strongly reduced ER and mitochondria co-localization (Fig. 7A). Moreover, the amount of fluorescence for mitochondria was significantly reduced in MYH9 and MYH10 knockdown cells. These results are consistent with that of LAMC2 knockdown cells (Fig. 5A). Western blotting analyses showed that knockdown of either MYH9 or MYH10 strongly induced GRP78 protein elevation, but reduced DRP1 and phosphorylated DRP1 protein levels (Fig. 7B), indicating that lack of MYH9 or MYH10 promoted ER stress and reduced ER-mitochondria interaction. Noted, knockdown of MYH9 reduced protein levels of MYH10, and vice versa. In addition, we silenced either MYH9 or MYH10 along with overexpression of LAMC2 to examine potential rescuing effects. Western blotting results showed that LAMC2 overexpression could partially offset the effects of si-MYH9 and si-MYH10 on GRP78 and DRP1 protein levels (Fig. 7C, D). These results further demonstrate that LAMC2 regulates ER-mitochondria interaction through MYH9 and MYH10. Furthermore, we showed that while LAMC2 overexpression promoted colony formation, knockdown of either MYH9 or MYH10 could counteract the oncogenic capacity of LAMC2 (Fig. 7E). Lastly, flow cytometry analyses showed that silencing either MYH9 or MYH10 effectively prevented LAMC2-induced increase in mitochondrial membrane potential (Fig. 7F), as well as decrease in ROS production (Fig. 7G). These results suggested that the lack of MYH9 or MYH10 prevents the ability for LAMC2 to inhibit ROS production and bolster mitochondrial function. Collectively, our data suggested that LAMC2 mitigates ER stress by promoting ER-mitochondria interaction by acting in complex with MYH9 and MYH10.

Fig. 7: LAMC2 enhances ER-mitochondria interaction and mitigates ER stress through binding to MYH9 and MYH10.

A Live-cell fluorescence images of co-localization between ER (green) and mitochondria (red) in MYH9 and MYH10 knockdown cancer cells. Quantification of ER/mitochondria co-localization (bottom). B Western blots of DRP1, MYH10, MYH9, GAPDH, and β-actin protein levels in MYH9 and MYH10 knockdown cancer cells. C Western blots of GRP78, DRP1, MYH10, MYH9, LAMC2, and GAPDH in co-transfected cancer cells. Cells were co-transfected with control, si-MYH9, si-MYH10, LAMC2, LAMC2+si-MYH9, or LAMC2+si-MYH10. D Western blots of GRP78, DRP1, MYH10, MYH9, LAMC2, and β-actin in co-transfected cancer cells. Cells were co-transfected with control, si-MYH9, si-MYH10, LAMC2, LAMC2+si-MYH9, or LAMC2+si-MYH10. E Colony formation of control and LAMC2 overexpressing cancer cells co-transfected with either si-MYH9 or si-MYH10. Relative clone number (right). F Flow cytometry analysis of JC1 (monomer %) in control and LAMC2 overexpressing cancer cells co-transfected with either si-MYH9 or si-MYH10 cancer cells. G Flow cytometry analysis of ROS levels in control and LAMC2 overexpressing cancer cells co-transfected with either si-MYH9 or si-MYH10 cancer cells. Graphical data were presented as mean ± SEM. *p < 0.05, **p < 0.01.

LAMC2 counteracts the effect of ER stress and promotes tumor growth in vivo

Prolonged ER stress and subsequent cellular apoptosis are detrimental for tumor growth. To examine the ability for LAMC2 to combat ER stress and promote tumor growth in vivo, we analyzed the real-time tumor growth in nude mice injected with either control or LAMC2 plasmid A549 cells treated with tunicamycin. As shown in Fig. 8A–D, xenografts in the LAMC2 group were significantly bigger than that of the control group. Moreover, while xenograft size, weight, and volume were significantly reduced through tunicamycin treatment in the control group, the effects were less significant in the LAMC2 group. In other words, LAMC2 overexpression efficiently impeded the tumor-inhibiting effect of tunicamycin. Immunohistochemistry staining of the tumor tissues showed that LAMC2 overexpression effectively reduced GRP78, and promoted Ki67 protein levels (Fig. 8E). These results strongly suggested that LAMC2 elicits tumor-promoting capacity by inhibiting ER stress. Interestingly, through bioinformatics analysis using the Cancer cell gene expression (CCLE) and profiling relative inhibition simultaneously in mixtures (PRISM) databases, we found that LAMC2 is positively correlated with seven chemotherapy drugs, including carboplatin, cytarabine, doxorubicin, teniposide, parbendazole, vincristine, and etoposide (Fig. 8F). Overall, as shown in Fig. 8G, results from our study provided compelling evidence that under ER stress, LAMC2 promotes tumor growth by enhancing protein complex formation with MYH9 and MYH10 to facilitate ER-mitochondria interaction, thereby mitigating ER stress-induced cell death.

Fig. 8: LAMC2 counteracts the effect of ER stress and promotes tumor growth in vivo.

A Bioluminescence imaging of xenograft at day 35 in BALB/c-nude mice injected with either control, LAMC2, control+Tun, or LAMC2+Tun A549 cells. B Image of extracted tumor tissue, demonstrating gross morphology and size. C Quantification of xenograft weight. D Quantification of xenograft volume. E IHC staining of LAMC2, GRP78, and Ki67 in tumor tissues. F Correlation between drug sensitivity and LAMC2 expression, analyzed using CCLE and AUC drug sensitivity data based on seven chemotherapeutic drugs downloaded from PRISM. G Graphical representation of increased ER-mitochondria interaction near the nucleus in response to ER stress and elevated LAMC2 levels. *p < 0.05, **p < 0.01.