Identification of the anticancer effects of a novel proteasome inhibitor, ixazomib, on colorectal cancer using a combined method of microarray and bioinformatics analysis

Figures & data

Figure 1 The inhibition of ixazomib on the proliferation of SW620 cells.

Figure 2 The alterations of gene expression profile.

Abbreviation: log₂FC, log₂fold change.

Table 1 The identified top 10 upregulated and downregulated DEGs between ixazomib-treated and untreated samples

Upregulated DEGs			Downregulated DEGs
Gene name	Log2FC	P-value	Gene name	Log2FC	P-value
KRT6A	6.2852	1.12E–07	APCDD1	−4.7126	1.69E–07
HSPA6	5.3542	1.42E–07	KRT40	−4.6060	4.98E–08
GABARAPL1	4.3530	1.86E–07	LINC01003	−4.2484	1.65E–07
ADM	4.2515	4.98E–08	FAM3B	−4.2399	1.40E–07
NCF2	4.2303	4.98E–08	LGR5	−3.9729	1.40E–07
MIR22HG	4.2234	3.05E–07	INHBE	−3.9095	0.000192
CD55	4.1233	4.98E–08	ASPSCR1	−3.8382	4.98E–08
DUSP5	4.0788	1.46E–07	TSTD1	−3.7015	4.98E–08
ZFAND2A	3.9474	6.17E–08	FGF3	−3.6730	1.01E–06
CXCL8	3.8177	5.91E–06	TFF3	−3.5758	5.78E–06

Abbreviations: DEGs, differentially expressed genes; log₂FC, log₂fold change.

Figure 3 Heatmap overview of the hierarchical clustering analysis.

Table 2 Top 35 predominant BP terms and all the eight CC terms and MF terms in GO functional enrichment analysis

Category	Term	Count	P-value
GO BP	GO:0006986~response to unfolded protein	9	5.85E–05
	GO:0007568~aging	16	3.79E–04
	GO:0046677~response to antibiotic	7	5.47E–04
	GO:0042981~regulation of apoptotic process	18	7.22E–04
	GO:0090200~positive regulation of release of cytochrome c from mitochondria	6	0.0020
	GO:0042149~cellular response to glucose starvation	6	0.0024
	GO:0007565~female pregnancy	10	0.0026
	GO:0042493~response to drug	21	0.0027
	GO:0090090~negative regulation of canonical Wnt signaling pathway	14	0.0030
	GO:0048661~positive regulation of smooth muscle cell proliferation	8	0.0035
	GO:0090084~negative regulation of inclusion body assembly	4	0.0036
	GO:0006950~response to stress	8	0.0038
	GO:0001666~response to hypoxia	14	0.0047
	GO:0042594~response to starvation	6	0.0055
	GO:2000483~negative regulation of interleukin-8 secretion	3	0.0062
	GO:0036499~PERK-mediated unfolded protein response	4	0.0063
	GO:1990440~positive regulation of transcription from RNA polymerase II promoter in response to endoplasmic reticulum stress	4	0.0063
	GO:0001836~release of cytochrome c from mitochondria	5	0.0063
	GO:0051090~regulation of sequence-specific DNA binding transcription factor activity	5	0.0085
	GO:0002009~morphogenesis of an epithelium	4	0.0099
	GO:0019626~short-chain fatty acid catabolic process	3	0.0102
	GO:0043065~positive regulation of apoptotic process	19	0.0109
	GO:0042026~protein refolding	4	0.0121
	GO:0006915~apoptotic process	30	0.0136
	GO:0050679~positive regulation of epithelial cell proliferation	7	0.0139
	GO:0072655~establishment of protein localization to mitochondrion	3	0.0149
	GO:0006977~DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest	7	0.0162
	GO:0097193~intrinsic apoptotic signaling pathway	5	0.0163
	GO:0044344~cellular response to fibroblast growth factor stimulus	5	0.0163
	GO:0044849~estrous cycle	4	0.0172
	GO:0032436~positive regulation of proteasomal ubiquitin-dependent protein catabolic process	7	0.0187
	GO:0042127~regulation of cell proliferation	13	0.0199
	GO:0032496~response to lipopolysaccharide	12	0.0202
	GO:0050873~brown fat cell differentiation	5	0.0203
	GO:0009408~response to heat	6	0.0205
GO-CC	GO:0005739~mitochondrion	83	7.17E–08
	GO:0070062~extracellular exosome	143	2.50E–07
	GO:0005759~mitochondrial matrix	31	7.53E–07
	GO:0005615~extracellular space	65	0.0034
	GO:0005777~peroxisome	10	0.0086
	GO:0000502~proteasome complex	7	0.0154
	GO:0022624~proteasome accessory complex	4	0.0183
	GO:0005829~cytosol	130	0.0402
GO-MF	GO:0005515~protein binding	339	4.72E–04
	GO:0000982~transcription factor activity, RNA polymerase II core promoter proximal region sequence-specific binding	5	0.0070
	GO:0016787~hydrolase activity	14	0.0138
	GO:0051087~chaperone binding	8	0.0202
	GO:0008046~axon guidance receptor activity	3	0.0216
	GO:0051082~unfolded protein binding	9	0.0340
	GO:0046703~natural killer cell lectin-like receptor binding	3	0.0355
	GO:0016791~phosphatase activity	5	0.0461

Note: Count refers to the number of DEGs enriched in the GO term.

Abbreviations: BP, biological process; CC, cellular component; DEGs, differentially expressed genes; GO, Gene Ontology; MF, molecular function; PERK, protein kinase R-like endoplasmic reticulum kinase.

Figure 4 The KEGG pathway of apoptosis.

Notes: The red-colored genes are differentially expressed genes enriched in the pathway in the study. Copyright ©2000. Reproduced from KEGG. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.²⁵
Abbreviations: ADP, adenosine diphosphate; CTLs, cytotoxic T lymphocytes; KEGG, Kyoto Encyclopedia of Genes and Genomes; MOMP, mitochondrial outer membrane permeabilization; NF-κB, nuclear factor-κB; NK, natural killer.

Table 3 The top 25 significantly enriched KEGG pathways of DEGs

ID	Term	Input number	Corrected P-value
hsa01100	Metabolic pathways	68	2.07E–12
hsa00280	Valine, leucine, and isoleucine degradation	11	9.53E–07
hsa04210	Apoptosis	13	0.0003
hsa00640	Propanoate metabolism	7	0.0003
hsa04115	p53 signaling pathway	9	0.0006
hsa00561	Glycerolipid metabolism	8	0.0011
hsa04010	MAPK signaling pathway	16	0.0012
hsa04146	Peroxisome	9	0.0012
hsa05169	Epstein–Barr virus infection	14	0.0012
hsa00052	Galactose metabolism	6	0.0012
hsa04110	Cell cycle	10	0.0038
hsa00650	Butanoate metabolism	5	0.0057
hsa04068	FoxO signaling pathway	10	0.0057
hsa04151	PI3K–Akt signaling pathway	17	0.0057
hsa01200	Carbon metabolism	9	0.0061
hsa04141	Protein processing in endoplasmic reticulum	11	0.0061
hsa04310	Wnt signaling pathway	10	0.0071
hsa00051	Fructose and mannose metabolism	5	0.0080
hsa04964	Proximal tubule bicarbonate reclamation	4	0.0174
hsa05132	Salmonella infection	7	0.0185
hsa05200	Pathways in cancer	17	0.0185
hsa03050	Proteasome	5	0.0212
hsa05205	Proteoglycans in cancer	11	0.0214
hsa00010	Glycolysis/gluconeogenesis	6	0.0214
hsa00520	Amino sugar and nucleotide sugar metabolism	5	0.0256

Note: Input number refers to the number of DEGs enriched in the pathway.

Abbreviations: DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5 Protein–protein interaction network.

Notes: Green nodes stand for downregulated genes, while red nodes stand for upregulated genes. The node size is positively correlated with the degree of the gene. The nodes’ colors from green to red reflect the log₂FC values of the genes from low to high.
Abbreviation: log₂FC, log₂fold change.

Table 4 Hub genes in the PPI network

Node	Degree	Node	Degree	Node	Degree	Node	Degree
TP53	84	IL8	22	TUBB2A	15	PSMD1	12
TSPO	65	HSPA6	19	SGK1	15	SYK	12
JUN	63	MYB	19	DUSP1	14	TNFSF10	11
FOS	49	CDC25A	18	HSPA2	14	ETS1	11
ITGA2	40	DNAJB1	17	DDIT3	14	PCCA	11
CDKN1A	33	GADD45A	17	RASSF1	14	CD44	11
DECR1	31	PSMD2	16	THNSL1	14	NMU	11
HSPA5	30	HSPH1	16	PSMD12	13	SERPINH1	11
BMP4	27	HADH	16	SFN	13	CA2	11
HMOX1	26	KRT16	16	ITPR1	13	BBS10	11
ATF3	24	FOSL1	15	HDAC4	13
ISG15	24	ANXA1	15	BAG3	13

Abbreviation: PPI, protein–protein interaction.

Figure 6 The significant modules in the protein–protein interaction network with MCODE score ≥5 and nodes ≥5.

Notes: (A) Module 1 with an MCODE score of 7.565 and the nodes of 24. (B) Module 2 with an MCODE score of 5 and the nodes of 9. (C) Module 3 with an MCODE score of 5 and the nodes of 5. Green nodes stand for downregulated genes, while red nodes stand for upregulated genes. The node size is positively correlated with the degree of the gene. The nodes’ colors from green to red reflect the log₂FC values of the genes from low to high.
Abbreviations: log₂FC, log₂fold change; MCODE, Molecular Complex Detection.

Table S1 The rest 32 BP terms in GO functional enrichment analysis

GO BP term	Count	P-value
GO:0007165~signal transduction	52	0.0223
GO:0055114~oxidation-reduction process	30	0.0226
GO:0009116~nucleoside metabolic process	4	0.0234
GO:0001822~kidney development	8	0.0234
GO:0071277~cellular response to calcium ion	6	0.0259
GO:0071493~cellular response to UV-B	3	0.0267
GO:0030178~negative regulation of Wnt signaling pathway	6	0.0279
GO:0071356~cellular response to tumor necrosis factor	9	0.0291
GO:0005975~carbohydrate metabolic process	12	0.0297
GO:0043407~negative regulation of MAP kinase activity	5	0.0300
GO:0071243~cellular response to arsenic-containing substance	3	0.0335
GO:0010165~response to X-ray	4	0.0345
GO:0000079~regulation of cyclin-dependent protein serine/threonine kinase activity	5	0.0388
GO:0006869~lipid transport	7	0.0393
GO:0008285~negative regulation of cell proliferation	21	0.0394
GO:0071456~cellular response to hypoxia	8	0.0394
GO:0003334~keratinocyte development	3	0.0410
GO:0070365~hepatocyte differentiation	3	0.0410
GO:0006105~succinate metabolic process	3	0.0410
GO:0031953~negative regulation of protein autophosphorylation	3	0.0410
GO:0006983~ER overload response	3	0.0410
GO:0045071~negative regulation of viral genome replication	5	0.0420
GO:0030819~positive regulation of cAMP biosynthetic process	5	0.0420
GO:0006974~cellular response to DNA damage stimulus	13	0.0434
GO:0007050~cell cycle arrest	10	0.0441
GO:0006954~inflammatory response	20	0.0470
GO:0030308~negative regulation of cell growth	9	0.0471
GO:0043433~negative regulation of sequence-specific DNA binding transcription factor activity	6	0.0475
GO:0030433~ER-associated ubiquitin-dependent protein catabolic process	6	0.0475
GO:0016266~O-glycan processing	6	0.0475
GO:0006935~chemotaxis	9	0.0490
GO:0006081~cellular aldehyde metabolic process	3	0.0491

Note: Count refers to the number of DEGs enriched in the GO term.

Abbreviations: BP, biological process; DEGs, differentially expressed genes; ER, endoplasmic reticulum; GO, Gene Ontology; MAP, mitogen-activated protein.

Table S2 The GO BP functional analysis of Module 1

GO BP term	Count	P-value
GO:0007565~female pregnancy	5	5.01E–06
GO:0042493~response to drug	6	3.85E–05
GO:0006366~transcription from RNA polymerase II promoter	7	3.88E–05
GO:0045944~positive regulation of transcription from RNA polymerase II promoter	8	1.78E–04
GO:1902895~positive regulation of pri-miRNA transcription from RNA polymerase II promoter	3	3.07E–04
GO:0045893~positive regulation of transcription, DNA-templated	6	4.55E–04
GO:0051090~regulation of sequence-specific DNA binding transcription factor activity	3	4.83E–04
GO:0043065~positive regulation of apoptotic process	5	5.67E–04
GO:0032570~response to progesterone	3	0.0012
GO:0007568~aging	4	0.0013
GO:0006954~inflammatory response	5	0.0014
GO:0008285~negative regulation of cell proliferation	5	0.0016
GO:0051591~response to cAMP	3	0.0016
GO:0042127~regulation of cell proliferation	4	0.0017
GO:0034097~response to cytokine	3	0.0021
GO:0061614~pri-miRNA transcription from RNA polymerase II promoter	2	0.0026
GO:0009612~response to mechanical stimulus	3	0.0027
GO:0048661~positive regulation of smooth muscle cell proliferation	3	0.0028
GO:0006950~response to stress	3	0.0029
GO:0060395~SMAD protein signal transduction	3	0.0030
GO:2000144~positive regulation of DNA-templated transcription, initiation	2	0.0091
GO:0009629~response to gravity	2	0.0104
GO:0008283~cell proliferation	4	0.0116
GO:0000733~DNA strand renaturation	2	0.0117
GO:0070365~hepatocyte differentiation	2	0.0130
GO:0032496~response to lipopolysaccharide	3	0.0193
GO:0035994~response to muscle stretch	2	0.0208
GO:0006940~regulation of smooth muscle contraction	2	0.0208
GO:0044849~estrous cycle	2	0.0221
GO:0030224~monocyte differentiation	2	0.0221
GO:0038095~Fc-epsilon receptor signaling pathway	3	0.0225
GO:0031668~cellular response to extracellular stimulus	2	0.0233
GO:0051412~response to corticosterone	2	0.0233
GO:0001701~in utero embryonic development	3	0.0246
GO:0045892~negative regulation of transcription, DNA-templated	4	0.0264
GO:0045987~positive regulation of smooth muscle contraction	2	0.0272
GO:0007186~G-protein coupled receptor signaling pathway	5	0.0276
GO:0010165~response to X-ray	2	0.0284
GO:0032967~positive regulation of collagen biosynthetic process	2	0.0297
GO:0045648~positive regulation of erythrocyte differentiation	2	0.0310
GO:0042981~regulation of apoptotic process	3	0.0313
GO:0021983~pituitary gland development	2	0.0361
GO:0006915~apoptotic process	4	0.0366
GO:2000379~positive regulation of reactive oxygen species metabolic process	2	0.0386
GO:0046677~response to antibiotic	2	0.0411
GO:0070555~response to interleukin-1	2	0.0424
GO:0010634~positive regulation of epithelial cell migration	2	0.0424
GO:0030501~positive regulation of bone mineralization	2	0.0449
GO:0045786~negative regulation of cell cycle	2	0.0474

Note: Count refers to the number of DEGs enriched in the GO BP term.

Abbreviations: BP, biological process; DEGs, differentially expressed genes; GO, Gene Ontology.

Table S3 The GO BP functional analysis of Module 2

GO BP term	Count	P-value
GO:0036499~PERK-mediated unfolded protein response	3	9.81E–06
GO:1990440~positive regulation of transcription from RNA polymerase II promoter in response to endoplasmic reticulum stress	3	9.81E–06
GO:0071850~mitotic cell cycle arrest	3	1.16E–05
GO:0007050~cell cycle arrest	3	0.0014
GO:0036500~ATF6-mediated unfolded protein response	2	0.0037
GO:0006983~ER overload response	2	0.0042
GO:0043065~positive regulation of apoptotic process	3	0.0063
GO:0034198~cellular response to amino acid starvation	2	0.0104
GO:2000379~positive regulation of reactive oxygen species metabolic process	2	0.0124
GO:0071479~cellular response to ionizing radiation	2	0.0129
GO:0043066~negative regulation of apoptotic process	3	0.0141
GO:0034605~cellular response to heat	2	0.0153
GO:0000079~regulation of cyclin-dependent protein serine/threonine kinase activity	2	0.0161
GO:0042542~response to hydrogen peroxide	2	0.0211
GO:0070373~negative regulation of ERK1 and ERK2 cascade	2	0.0239
GO:0043433~negative regulation of sequence-specific DNA binding transcription factor activity	2	0.0247
GO:0006977~DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest	2	0.0256
GO:0006979~response to oxidative stress	2	0.0450

Note: Count refers to the number of DEGs enriched in the GO BP term.

Abbreviations: ATF6, activating transcription factor 6; BP, biological process; DEGs, differentially expressed genes; ER, endoplasmic reticulum; ERK1, extracellular signal-regulated kinases 1; ERK2, extracellular signal-regulated kinases 2; GO, Gene Ontology; PERK, protein kinase R-like endoplasmic reticulum kinase.

Table S4 The GO BP functional analysis of Module 3

GO BP term	Count	P-value
GO:0090090~negative regulation of canonical Wnt signaling pathway	5	8.56E–09
GO:0006521~regulation of cellular amino acid metabolic process	4	1.05E–07
GO:0002479~antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent	4	2.01E–07
GO:0038061~NIK/NF-kappaB signaling	4	2.31E–07
GO:0051436~negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle	4	2.89E–07
GO:0051437~positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition	4	3.55E–07
GO:0031145~anaphase-promoting complex-dependent catabolic process	4	4.00E–07
GO:0060071~Wnt signaling pathway, planar cell polarity pathway	4	6.34E–07
GO:0043488~regulation of mRNA stability	4	8.93E–07
GO:0002223~stimulatory C-type lectin receptor signaling pathway	4	9.46E–07
GO:0033209~tumor necrosis factor-mediated signaling pathway	4	1.35E–06
GO:0090263~positive regulation of canonical Wnt signaling pathway	4	1.42E–06
GO:0050852~T-cell receptor signaling pathway	4	2.67E–06
GO:0038095~Fc-epsilon receptor signaling pathway	4	4.65E–06
GO:0000209~protein polyubiquitination	4	5.14E–06
GO:0043161~proteasome-mediated ubiquitin-dependent protein catabolic process	4	6.90E–06
GO:0000165~MAPK cascade	4	1.48E–05

Note: Count refers to the number of DEGs enriched in the GO BP term.

Abbreviations: BP, biological process; DEGs, differentially expressed genes; GO, Gene Ontology; MAPK, mitogen-activated protein kinase; MHC, major histocompatibility complex; NF-kappaB, nuclear factor-kappaB; NIK, nuclear factor-kappaB-inducing kinase; TAP, transporter associated with antigen processing.

KanehisaMGotoSKEGG: kyoto encyclopedia of genes and genomesNucleic Acids Res2000281273010592173

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