Cross-cancer profiling of molecular alterations within the human autophagy interaction network

Figures & data

Figure 1. Network representation of core autophagy proteins, autophagy regulators and pathway interactors, clustered into functional modules of human protein-protein interactions by the Reactome Functional Interaction Network. Schematic showing the functional interconnectedness of 162 of 211 autophagy-associated proteins curated from literature. Edges represent human protein-protein interactions (PPI) reported in the Reactome database. Cellular functional compartments that are important in autophagy modulation are indicated by color-coded modules annotated by module-enriched GO Biological Process terms (FDR < 0.01). Node size ranks proteins by PPI count.

Figure 2. Circos plot overview of mutation frequency for significantly mutated autophagy-associated genes found in patients in 6 different cancers. Ribbons connect cancer type to mutated gene, ribbon color denotes levels of mutation frequency (range 0.012–0.176). Mutated genes were found to be under positive selection for somatic single nucleotide mutation, as assessed by an analysis of patterns of Darwinian selective pressure (adjusted P < 0.05). UCEC, uterine corpus endometrial carcinoma; LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; GBM, glioblastoma multiforme.

Figure 3 (See previous page). Differential gene expression analysis of pooled tumor versus pooled matched normal tissue gene abundance for 97 invasive breast carcinomas (BRCA) and 65 clear cell renal carcinomas (KIRC). (Right) Boxplots of normalized RNA-seq derived gene abundance (RPKM) displayed for autophagy-associated genes differentially expressed in tumor tissue compared to adjacent matched normal tissue in BRCA and KIRC patients. Differentially expressed genes showed significant (adjusted P < 0.05) differences in mRNA levels with a median fold-change > 2. (Left) Unsupervised clustering on only differentially expressed autophagy-associated genes (log2 transformed RPKM) grouped matched tumor and normal samples together.

Figure 4. Differential expression analysis of pooled tumor versus pooled matched normal gene abundance for 17 lung squamous cell carcinomas (LUAD) and 25 lung adenocarcinomas (LUSC). (Right) Boxplots of normalized RNA-seq derived gene abundance (RPKM) displayed for autophagy-associated genes differentially expressed in tumor tissue compared to adjacent matched normal tissue in LUAD and LUSC patients. Differentially expressed genes showed significant (adjusted P < 0.05) differences in mRNA levels with a median fold-change > 2. (Left) Unsupervised clustering on only differentially expressed autophagy-associated (log2 transformed RPKM) genes grouped matched tumor and normal samples together.

Table 1. Summary of autophagy-associated genes found to be differentially expressed between tumor and matched normal tissue, in 2 or more cancer types

Gene	Putative autophagy association	BRCA	KIRC	LUAD	LUSC
CDK5R1	Positive modulator^*	↑	↑	↑	↑
CDKN2A	Positive modulator	↑	↑	↑	↑
BNIP3	Positive modulator, mitophagy	—	↑	↑	↑
DIRAS3	Positive modulator	—	↓	↓	↓
GABARAPL1	Core gene	↓	↓	↓	—
LAPTM4B	Positive modulator	—	↓	↑	↑
LRRK2	Negative modulator	↓	—	↓	↓
MAP1LC3C	Core gene	↓	—	↓	↓
PPP2R2C	Modulator, direction undefined	↑	—	↑	↑
PRKAR2B	Positive modulator	↓	↓	—	↓
DNM1	Positive modulator, mitophagy	—	↑	—	↑
DRAM1	Positive modulator	—	—	↓	↓
PIK3C2G	Modulator, direction undefined	↓	↓	—	—
PPP2R2B	Modulator, direction undefined	↓	↓	—	—
SNX30	Human homolog of yeast SNX4/ATG24	—	—	↓	↓
STBD1	GABARAPL1 interactor	↓	—	—	↓
TRAF2	Positive modulator	↑	↑	—	—

* Modulator is defined in this study as an autophagy pathway interactor reported in literature to be associated with autophagy modulation that lacks a defined direction of regulation.

BRCA, invasive breast carcinoma; KIRC, kidney clear cell renal cell carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; ↑, mRNA significantly upregulated compared to matched normal tissue; ↓, mRNA significantly downregulated compared to matched normal tissue; —, not differentially expressed.

Table 2. Summary of autophagy-associated genes found to have significantly increased or decreased mRNA in patients with disease-specific molecular alterations or clinical phenotypes (previously reported to be key in literature or identified in this study), compared to patients not harboring those alterations or phenotypes, in 7 cancer types

Gene	BRCA	HNSC	KIRC	LAML	LUAD	LUSC	UCEC
ANKFY1	—	—	—	FAB M3 ↑PML-RARA ↑t(15;17) ↑	—	—	—
ATG3	—	—	—	FAB M5 ↑	—	—	—
BNIP3	—	HPV positive ↓	—	—	—	KEAP1 mut ↑MTOR mut ↑SMARCA4 mut ↑	—
C12orf44	—	—	—	—	—	—	ATG4C mut ↑
CALCOCO2	—	—	—	Del 5q ↓inv(16) ↑TP53 mut ↓	—	—	—
CDKN2A	Basal-like↑	HPV positive ↑Tongue base ↑Tonsil ↑TP53 mut ↓	—	—	—	RB1 mut ↑	Endometrioid ↓Serous ↑Stage I ↓Stage III ↑Tumor G2 ↓Tumor G3 ↑
CEBPB	Basal-like↑BRCA1 mut ↑	—	—	FAB M5 ↑	—	—	Endometrioid ↓Serous ↑
CLN3	FGFR2 amp ↑	—	—	—	—	—	—
COPZ1	Basal-like↑	—	—	—	—	—	—
DNM1L	KRAS amp ↑	—	—	—	—	—	—
GABARAP	—	—	—	Del 5q ↓FAB M0 ↓FAB M4 ↑TP53 mut ↓	—	—	Endometrioid ↑Serous ↓
GABARAPL1	—	—	PTEN mut ↓	—	—	—	—
GABARAPL2	—	—	—	—	—	AKT2 amp ↑	—
GBAS	—	EGFR amp ↑EGFR muts ↑	—	—	—	EGFR amp ↑	—
HADHA	FGFR2 amp ↑	—	—	—	—	—	—
HIF1A	KRAS amp ↑	HPV positive ↓Tonsil ↓	—	—	—	—	—
KEAP1	—	—	—	—	—	NFE2L2 mut ↑	—
LAMP1	—	HPV positive ↓	—	—	—	—	—
LAPTM4B	AKT1 mut ↓Basal-like↑BRCA1 mut ↑BRCA2 mut ↑CDH1 mut ↓IGF1R amp ↑KMT2C mut ↓Luminal A ↓MAP2K4 mut ↓MAP3K1 mut ↓MYC amp ↑NBN amp ↑TP53 mut ↑	HRAS mut ↓	—	Del 5q ↑Del 7q ↑Poor molecular risk ↑TP53 mut ↑	CDKN2A homdel↓Lymphnode spread n0 ↑	—	—
NFE2L2	—	NFE2L2 amp ↑NFE2L2 mut ↑SOX2 amp ↑	—	—	—	EGFR amp ↑SOX2 amp ↑TP53 mut ↑	—
PTPRS	—	—	—	—	—	—	ATG4C mut ↑
RAB5B	Basal-like ↓	—	—	FAB M3 ↑Good molecular risk ↑PML-RARA ↑t(15;17) ↑	—	—	—
RAB5C	Basal-like ↓	—	—	—	—	—	—
SQSTM1	KRAS amp ↓	NFE2L2 mut ↑ Tongue base ↑	—	—	KEAP1 mut ↑	NFE2L2 mut ↑	NFE2L2 mut ↑
TAB2	—	—	—	FAB M0 ↑	—	—	—
TNFSF10	PIK3CA amp ↑	HPV positive ↑Larynx ↓	PTEN mut ↓	—	KEAP1 mut ↓STK11 mut ↓	FGFR1 amp	ATG4C mut ↓BIRC6 mut ↓CDK5RAP2 mut ↓Endometrioid ↓Serous ↑LRRK2 mut ↓MAPK8 mut ↓MTMR7 mut ↓PRKACG mut ↓TAB3 mut ↓
VCP	—	—	—	—	—	—	ATG4C mut ↑
WDR45L	—	—	—	—	SMARCA4 mut ↑	—	—
YKT6	—	HPV positive ↓	—	—	—	—	—

↑, significantly increased AA mRNA; ↓, significantly decreased mRNA; amp, amplification; homdel, homozygous deletion; mut, mutant; BRCA, invasive breast carcinoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney clear cell renal cell carcinoma; LAML, acute myeloid leukemia; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; UCEC, uterine corpus endometrial carcinoma; Tumor G2, tumor grade 2; Tumor G3, tumor grade 3.

Figure 5 (See previous page). Unsupervised consensus clustering of autophagy-associated tumor gene abundance stratified patient overall survival for acute myeloid leukemia (LAML), kidney clear cell renal carcinoma (KIRC), and head and neck squamous cell carcinoma (HNSC), linking differential expression of autophagy regulators and interactors to enriched clinical features within patient groups. Unsupervised consensus clustering by non-negative matrix factorization (NMF) on mRNA levels of 211 autophagy-associated genes stratified patients into groups of significantly different overall survival (Log rank P < 0.05). Kaplan-Meier curves of overall survival are shown for HNSC, KIRC, and LAML. Disease-specific clinical covariates (diagnostic, prognostic and molecular phenotypes) found to be enriched within overall survival (OS) groups following clustering (Fisher exact test P < 0.05) are drawn below respective cancer types. Patient status for each covariate was obtained from clinical metadata collected by TCGA. Patient counts (n) for each covariate are noted. Enrichment is depicted as color-coded distributions of the relative frequency of each feature across OS groups per cancer type. Differentially expressed autophagy-associated genes, identified within individual OS groups compared to all other OS groups combined (Wilcoxon rank sum test P < 0.05; median fold-change > 1.5), are reported per cancer type. Boxplots of normalized RNA-seq derived gene abundance (RPKM) of differentially expressed genes are drawn for each OS group.

Figure 6 (See previous page). Summary by gene of molecular alterations found to target autophagy-associated genes across multiple cancer types. This overview includes all significant somatic point mutation and differential gene expression identified in autophagy-associated genes in this study. Significant results were obtained from a differential expression analysis of tumor versus matched adjacent normal tissue for 4 cancer types examined (BRCA, KIRC, LUAD, and LUSC; detailed in Figs. 3 and 4), as well as from a differential expression analysis between patient groups showing significantly different overall survival for 3 of 9 cancer types (HNSC, KIRC and LAML; detailed in Fig. 5). Rows represent autophagy-associated genes found to have at least one significant alteration (either mutation or differential expression) in at least one context investigated. Groups of columns describe a single, major analysis in this study. The first column reports genes found to be significantly mutated in any of 6 of 11 cancer types investigated (detailed in Fig. 2), with annotation of cancer type and mutation frequency found to the left. Cancer type, relative overall survival (OS) of patient groups, and value for differential expression of mRNA are color-coded, as described in the legend.