Plectin-mediated cytoskeletal crosstalk as a target for inhibition of hepatocellular carcinoma growth and metastasis

Reviewer #1 (Public review):

Summary:

This study investigated the role of plectin, a cytoskeletal crosslinker protein, in liver cancer formation and progression. Using the liver-specific Plectin knockout mouse model, the authors convincingly showed that PLECTIN is critical for hepatocarcinogenesis, as functional inhibition of plectin suppressed tumor formation in several models. They also provided evidence to show that inhibition of plectin inhibited HCC cell invasion and reduced metastatic outgrowth in the lung. Mechanistically, they suggested that plectin inhibition attenuated FAK, MAPK/ERK, and PI3K/AKT signaling.

Strengths:

The authors generated a liver-specific plectin knockout mouse model. By using DEN and sgP53/MYC models, the authors convincingly demonstrated an oncogenic role of PLECTIN in HCC development. plecstatin-1 (PST), as a plectin inhibitor, showed promising efficacy in inhibiting HCC growth, which provides a basis for potentially treating HCC using PST.

The MIR images for tracking tumor growth in animal models were compelling. The high-quality confocal images and related qualifications convincingly showed the impact of plectin functional inhibition on contractility and adhesions in HCC cells.

Weaknesses:

The conclusions of this paper are primarily well supported by data. However, some claims were not fully supported by the data presented.

The authors suggest that plectin controls oncogenic FAK, MAPK/Erk, and PI3K/Akt signaling in HCC cells, representing the mechanisms by which plectin promotes HCC formation and progression. However, the effect of plectin inactivation on these signaling was inconsistent in Huh7 and SNU-475 cells (Figure 3D), despite similar cell growth inhibition in both cell lines (Figure 2G). For example, pAKT and pERK were only reduced by plectin inhibition in SNU-475 cells but not in Huh7 cells. In addition, pFAK was not changed by plectin inhibition in both cells, and the ratio of pFAK/FAK was increased in both cells. Thus, it is hard to convince me that plectin promotes HCC formation and progression by regulating these signalings. Overall, the mechanistic studies in this manuscript lack sufficient depth.

The authors claimed that plectin inactivation inhibits HCC invasion and metastasis using in vitro and in vivo models. However, the results from in vivo models were not as compelling as the in vitro data. The lung colonization assay is not an ideal in vivo model for studying HCC metastasis and invasion, especially when plectin inhibition suppresses HCC cell growth and survival. Using an orthotopic model that can metastasize into the lung or spleen could be much more convincing for an essential claim. Also, in Figure 6H, histology images of lungs from this experiment need to be shown to understand plectin's effect on metastasis better. Figure 6G, it is unclear how many mice were used for this experiment. Did these mice die due to the tumor burdens in the lungs?

The whole paper used inhibition strategies to understand the function of plectin. However, the expression of plectin in Huh7 cells is low (Figure 1D). It might be more appropriate to overexpress plectin in this cell line or others with low plectin expression to examine the effect on HCC cell growth and migration.

Reviewer #2 (Public review):

Summary:

Plectin is a cytolinker that associates with all three main components of the cytoskeleton and intercellular junctions and is essential for epithelial tissue integrity. Previous reports showed that PLEC regulates tumor growth and metastasis in different cancers. In this manuscript, the authors described PLEC as a target in the initiation and growth of HCC. They showed that inhibiting PLEC reduced tumorigenesis in different in vitro and in vivo HCC models, including in a xenograft model, DEN model, oncogene-induced HCC model, and a lung metastasis model. Mechanistically, the authors showed that inhibiting PLEC results in a disorganized cytoskeleton, deficiency in cell migration, and changes in relevant signaling pathways.

Strengths:

In general, the data are shown in multiple ways and support the main conclusion of the manuscript. The results add to the field by highlighting the importance of cellular mechanics in cancer progression.

Weaknesses:

(1) The annotation of mouse numbers is confusing. In Figures 2A B D E F, it should be the same experiment, but the N numbers in A are 6 and 5. In E and F they are 8 and 3. Similarly, in Figure 2H, in the tumor size curve, the N values are 4,4,5,6. In the table, N values are 8,8,10,11 (the authors showed 8,7,8,7 tumors that formed in the picture).

(2) In Figure 3D and Figure S3C, the changes in most of the proteins/phosphorylation sites are not convincing/consistent. These data are not essential for the conclusion of the paper and WB is semi-quantitative. Maybe including more plots of the proteins from proteomic data could strengthen their detailed conclusions about the link between Plectin and the FAK, MAPK/Erk, PI3K/Akt pathways as shown in 3E.

(3) Figure S7A and B, The pictures do not show any tumor, which is different from Figure 7A and B (and from the quantification in S7A lower right). Is it just because male mice were used in Figure 7 and female mice were used in Figure S7? Is there literature supporting the sex difference for the Myc-sgP53 model?

(4) Figure 2F, S2A, PleΔAlb mice more frequently formed larger tumors, as reflected by overall tumor size increase. The interpretation of the authors is "possibly implying reduced migration or increased cohesion of plectin-depleted cells". It is quite arbitrary to make this suggestion in the absence of substantial data or literature to support this theory.

(5) Mutation or KO PLEC has been shown to cause severe diseases in humans and mice, including skin blistering, muscular dystrophy, and progressive familial intrahepatic cholestasis. Please elaborate on the potential side effects of targeting plectin to treat HCC.

Reviewer #3 (Public review):

Summary:

In this manuscript, Outla Z et al described the analysis of plectin in HCC pathogenesis. Specifically, it was found that elevated plectin levels in liver tumors, correlated with poor prognosis for HCC patients. Mechanistically, it showed that plectin-dependent disruption of cytoskeletal networks leads to the attenuation of oncogenic FAK, MAPK/Erk, and PI3K/AKT signals. Finally, the authors showed that plectin inhibitor plecstatin-1 (PST) is well-tolerated and capable of overcoming therapy resistance in HCC.

Strengths:

The studies of plectin are not entirely novel (Pubmed: 36613521). Nevertheless, the current manuscript provides a much more detailed mechanistic study and the results have translational implications. Additional strengths include convincing cell biology data, such as plectin regulates cytoskeletal networks, and HCC migration/invasion.

Weaknesses:

Multiple major issues are noted, and the conclusion is not well supported by the data presented.

(1) The rationale for using Huh7 cells in the manuscript is not well explained as it has the lowest plectin expression levels.

(2) The KO cell experiments should be supplemented with overexpression experiments.

(3) There is significant concern that while ablation of Ple led to reduced tumor number, these mice had larger tumors. The data indicate that plectin may have distinct roles in HCC initiation versus progression. The data are not well explained and do not fully support that plectin promotes hepatocarcinogenesis.

(4) Figure 3 showed that plectin does not regulate p-FAK/FAK expression. Therefore, the statement that plectin regulates the FAK pathway is not valid. Furthermore, there are too many variables in turns of p-AKT and p-ERK expression, making the conclusion not well supported.

(5) The studies of plecstatin-1 in HCC should be expanded to a panel of human HCC cells with various plectin expression levels in turns of cell growth and cell migration. The IC50 values should be determined and correlate with plectin expression.

(6) One of the major issues is the mechanistic studies focusing on plectin regulating HCC migration/metastasis, whereas the in vivo mouse studies focus on HCC formation (Figures 3 and 7). These are distinct processes and should not be mixed.

(7) Figure 7B showed that Ple KO mice were treated with PST, but the data are not presented in the manuscript. Tumor cell proliferation and apoptosis rates should be analyzed as well.

(8) The status of FAK, AKT, and ERK pathway activation was not analyzed in mouse liver samples. In Figure 7D, most of the adjusted p-values are not significant.

(9) There is no evidence to support that PST is capable of overcoming therapy resistance in HCC. For example, no comparison with the current standard care was provided in the preclinical studies.