LRP1B suppresses HCC progression through the NCSTN/PI3K/AKT signaling axis and affects doxorubicin resistance

Abstract

Accumulating evidence supports the association of somatic mutations with tumor occurrence and development. We aimed to identify somatic mutations with important implications in hepatocellular carcinoma (HCC) and explore their possible mechanisms. The gene mutation profiles of HCC patients were assessed, and the tumor mutation burden was calculated. Gene mutations closely associated with tumor mutation burden and patient overall survival were identified. In vivo and in vitro experiments were performed to verify the effects of putative genes on proliferation, invasion, drug resistance, and other malignant biological behaviors of tumor cells. Fourteen genes with a high mutation frequency were identified. The mutation status of 12 of these genes was closely related to the mutation burden. Among these 12 genes, LRP1B mutation was closely associated with patient prognosis. Nine genes were associated with immune cell infiltration. The results of in vivo and in vitro experiments showed that the knockdown of LRP1B promotes tumor cell proliferation and migration and enhances the resistance of tumor cells to liposomal doxorubicin. LRP1B could directly bind to NCSTN and affect its protein expression level, thereby regulating the PI3K/AKT pathway. Our mutational analysis revealed complex and orchestrated liposomal alterations linked to doxorubicin resistance that may also render cancers less susceptible to immunotherapy and also provides new treatment alternatives.

Keywords: Doxorubicin; Hepatocellular carcinoma; LRP1B; PI3K/AKT pathway; Tumor mutation burden.

Figure 1

Gene mutation frequency statistics and screening of tumor mutation burden-related mutations. (A) Gene mutation frequencies in the TCGA database. (B) Gene mutation frequencies in the International Cancer Genome Consortium (ICGC) database. (C) Waterfall plot of gene mutation statuses in The Cancer Genome Atlas (TCGA) database. (D) Waterfall plot of gene mutation statuses in the ICGC database. (E) Venn diagram of mutated genes. (F) Mutation frequencies of the overlapping genes. (G) Correlation analysis of TMB and gene mutations.

Figure 2

Prognostic analysis and immune cell infiltration analysis. (A–N) Survival curves of patients with mutant and wild-type genes: XIRP2 (A), PCLO (B), OBSCN (C), MUC16 (D), USH2A (E), LRP1B (F), HMCN1 (G), FLG (H), TP53 (I), CSMD3 (J), APOB (K), AHNAK2 (L), TTN (M) and ADGRV1 (N). (O) Univariate Cox regression analysis. (P) Multivariate Cox regression analysis. (Q) Differential analysis of immune cell infiltration based on LRP1B mutation status (mutant or wild-type). (R) Gene set enrichment analyses (GSEA) based on LRP1B mutation status.

Figure 3

LRP1B mutation-related DEGs and functional enrichment analysis. (A–J) Expression levels of f106 (A), SCN1B (B), DNASE1L3 (C), APTR (D), TCF19 (E), LOC101927021 (F), TMEM14A (G), TMEM97 (H), LOC730101 (I) and LIMD1 (J) in the LRP1B mutant and wild-type groups. (K–O) Molecular structure diagrams of serdemetan (K), alisertib (L), caffeic acid (M), pyrimethamine (N), and doxorubicin (O). (P) Gene ontology (GO) enrichment analysis. (Q) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

Figure 4

Low expression of LRP1B in hepatocellular carcinoma is associated with cell migration, proliferation, and apoptosis. (A)LRP1B expression was measured in adjacent and tumor tissues. (B) Relative mRNA expression of LRP1B in different cell lines. (C) Immunofluorescence staining for LRP1B. (D) EdU incorporation assay. (E) Scratch assay of Huh7 cells. (F) Plate colony formation assay. (G) Transwell migration assay. (H) Apoptosis was analyzed by flow cytometry. (I) The cell cycle distribution was analyzed by flow cytometry.

Figure 5

Effects of LRP1B on tumor progression and doxorubicin resistance. (A) Plate clone formation assay. (B) Scratch assay of HCCLM3 cells. (C) Lung metastatic foci in the lung metastasis model. (D) Photographs and growth curves of subcutaneous xenografts. (E) Immunohistochemical staining for the expression of LRP1B and Ki67 in subcutaneous xenograft tissues. (F) IC₅₀ values in different cell lines were determined based on 50% growth inhibition using a CCK-8 assay. (G) IC₅₀ values were determined in LRP1B knockdown Huh7 cells and control cells. (H) Growth curves of subcutaneous xenografts treated with doxorubicin. (I) Detection of apoptotic Huh7 cells after doxorubicin and shLRP1B treatment.

Figure 6

LRP1B directly binds to NCSTN and regulates the PI3K/AKT/mTOR pathway. (A)LRP1B knockdown causes upregulation of the PI3K/AKT pathway. (B)LRP1B overexpression inhibited the PI3K/AKT pathway. (C)LRP1B regulates the PI3K/AKT/mTOR pathway. (D) Immunoprecipitation (IP) assay in Huh7 cells. (E) IP assay in 293 T cells using a primary antibody specific for HA. (F) IP was performed with a primary antibody specific for Flag.

Figure 7

LRP1B regulates the ubiquitination, degradation, and protein level of NCSTN. (A) The rate of NCSTN degradation after cycloheximide (CHX) inhibition of protein synthesis. (B)NCSTN protein levels were determined after MG132 treatment. (C) The proteasome inhibitor MG132 restored the LRP1B-mediated reduction in the NCSTN protein level in cancer cells. (D)LRP1B regulates the ubiquitination of NCSTN.

Figure 8

LRP1B regulates the malignant behavior of tumor cells through the PI3K/AKT/mTOR pathway. (A) Proliferation was evaluated by a CCK-8 assay. (B) Plate colony formation assays were used to detect cell proliferation. (C) Scratch assay. (D) Flow cytometry was used to detect apoptosis in transfected cells. (E) Subcutaneous xenografts in mice from the indicated groups. (F, G) Immunohistochemical staining for LRP1B (F) and NCSTN (G) in subcutaneous xenograft tissues. (H) Immunohistochemical staining for Ki-67. (I) Oil Red O staining of subcutaneous xenograft tissues.

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