DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kDa GTPase with 60% amino acid homology to RAS, but with a distinctive 34 amino acid N-terminal extension required to block RAS function. DIRAS3 is maternally imprinted and expressed only from the paternal allele in normal cells. Loss of expression can occur in a single “hit” through multiple mechanisms. Downregulation of DIRAS3 occurs in cancers of the ovary, breast, lung, prostate, colon, brain, and thyroid. Reexpression of DIRAS3 inhibits signaling through PI3 kinase/AKT, JAK/STAT, and RAS/MAPK, blocking malignant transformation, inhibiting cancer cell growth and motility, and preventing angiogenesis. DIRAS3 is a unique endogenous RAS inhibitor that binds directly to RAS, disrupting RAS dimers and clusters, and preventing RAS-induced transformation. DIRAS3 is essential for autophagy and triggers this process through multiple mechanisms. Reexpression of DIRAS3 induces dormancy in a nu/nu mouse xenograft model of ovarian cancer, inhibiting cancer cell growth and angiogenesis. DIRAS3-mediated induction of autophagy facilitates the survival of dormant cancer cells in a nutrient-poor environment. DIRAS3 expression in dormant, drug-resistant autophagic cancer cells can serve as a biomarker and as a target for novel therapy to eliminate the residual disease that remains after conventional therapy.

DIRAS3 [DIRAS family GTPase 3, also known as Aplasia Ras Homology I (ARHI) and Normal Ovarian Epithelium Y2 (NOEY2)] is a maternally imprinted tumor suppressor gene that was first found to be downregulated in ovarian cancers by our group (1). DIRAS3, a member of the RAS superfamily, maps to human chromosome 1p31.3 and encodes a 26 kDa GTPase with 60% amino acid homology to Ras, but differs from RAS by the addition of a distinctive 34 amino acid N-terminal extension (NTE) (2). This NTE region is required for DIRAS3 to reverse RAS function and to act as a tumor suppressor. DIRAS3 is maternally imprinted and expressed only from the paternal allele in all normal cells. Downregulation of DIRAS3 expression can occur genetically through loss of heterozygosity (LOH), epigenetically by methylation of both the imprinted and nonimprinted alleles, transcriptionally by E2F1 and E2F4, or posttranscriptionally by miR221 or miR222 and by decreased association with HuR RNA-binding proteins (3–8). DIRAS3 is downregulated not only in ovarian cancer, but also in cancers of the breast, lung, prostate, colon, brain, and thyroid, consistent with its general importance for carcinogenesis (Table 1).

Table 1.

Role of DIRAS3 in different cancers.

TypeMechanism of inactivation (compared with adjacent normal tissues)Effect of DIRAS3REFERENCES
Ovarian cancer Hypermethylation • Loss of expression linked to tumor progression (1, 10) 
 LOH • Shortened disease-free survival and reduced expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (31) 
 Transcriptional repression • Reexpression induces autophagy, blocks the PI3K pathway, induces dormancy in in vivo models (50, 56) 
 Posttranscriptional repression • Downregulation increases cell migration through activation of the Stat3 and FAK/Rho signaling pathways (38, 44) 
 miRNA • Reexpression decreases cellular ATP/ADP, increases oxidative stress, and decreases mitochondrial function. (74) 
Breast cancer Hypermethylation • Dramatic downregulation observed in more than 70% of breast carcinomas (1, 5, 10) 
 LOH   
 Transcriptional repression • Loss of expression has been linked to tumor progression from DCIS to invasive carcinoma (39) 
 Posttranscriptional repression • When reexpressed, enhances growth inhibitory effect by promoting autophagy, apoptosis, and G2–M cell cycle arrest (75) 
 miRNA • DIRAS3 repression by JMJD2A promotes breast cancer progression (76) 
Pancreatic cancer Deletion • DIRAS3 expression is downregulated in pancreatic cancer (48%) compared with normal pancreatic tissue (18%), plays a role as tumor suppressor gene (77) 
 Hypermethylation • Reexpression of DIRAS3 in PDAC cell lines blocks cell cycle progression at G1 phase by inhibiting PI3K/AKT and NF-κB signaling pathways in addition to inhibiting ERK activity (33, 57) 
Thyroid cancer Deletion • A key early event observed in 85% of follicular thyroid cancers (23) 
 Hypermethylation • Advanced pathologic stage, poor differentiation degree, and worse prognosis, lower 3-year survival rates (78) 
Colon cancer Downregulation • Advanced tumor grade (79) 
 LOH   
 Promoter methylation • Reexpression of DIRAS3 inhibits cells invasion and adhesion through suppressing EMT via the suppression of the Wnt/β-catenin signaling pathway (80) 
Esophageal carcinoma Deletion • Activation of the Wnt pathway, decreased overall survival, increased invasive capacity, cell cycle progression and epithelial-mesenchymal transition (30) 
 Downregulation   
HCC Promoter hypermethylation Somatic mutation • Tumor growth, angiogenesis, impaired apoptosis, hepatocarcinogenesis (54) 
Lung cancer Hypermethylation • Aberrant methylation profile compared with normal tissue (81) 
 Downregulation • Impaired cyclin D1, MMP-1, and MMP-2 expression, deregulated cancer cell growth and proliferation, invasion, impaired apoptosis (82) 
Multiple myeloma Downregulation • Downregulation results in over-angiogenic phenotype (53) 
Osteosarcoma Downregulation • Development of osteosarcoma formation, promotes cell proliferation by enhancing PI3K/AKT signaling, attenuates apoptosis (83) 
 Hypermethylation • Zebularine, a demethylating agent repressing DNMT1, prevents methylation of DIRAS3 and increase its expression, inhibits the viability, and promotes apoptosis in osteosarcoma cells. (84) 
  • Reduced GAS5 lncRNA and increased miR-221.  
  • Overexpression of GAS5 suppresses the proliferation, migration, and EMT of osteosarcoma cells by enhancing DIRAS3 expression, suppresses tumor volume, Ki-67 and PCNA staining (85) 
Prostate cancer Downregulation • Overexpression inhibits cell proliferation, colony formation, invasion, and induces apoptosis (86) 
 Hypermethylation   
 Targeting of 3′UTR of DIRAS3 by miR221/222 • Lower DIRAS3 and higher miRNA–221/222 levels correlate with more aggressive prognosis, suggested as a biomarker for disease progression (4) 
Renal cancer Decreased mRNA levels • Advanced tumor stage, distant metastasis phenotype, lower survival rate (87) 
SCC Aberrant methylation • Development and progression of tongue SCC (88) 
  • Concurrent reexpression of p53 and DIRAS3 effectively induces cell death and decreases the tumor volume in HNSCC xenograft mouse model (89) 
TypeMechanism of inactivation (compared with adjacent normal tissues)Effect of DIRAS3REFERENCES
Ovarian cancer Hypermethylation • Loss of expression linked to tumor progression (1, 10) 
 LOH • Shortened disease-free survival and reduced expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (31) 
 Transcriptional repression • Reexpression induces autophagy, blocks the PI3K pathway, induces dormancy in in vivo models (50, 56) 
 Posttranscriptional repression • Downregulation increases cell migration through activation of the Stat3 and FAK/Rho signaling pathways (38, 44) 
 miRNA • Reexpression decreases cellular ATP/ADP, increases oxidative stress, and decreases mitochondrial function. (74) 
Breast cancer Hypermethylation • Dramatic downregulation observed in more than 70% of breast carcinomas (1, 5, 10) 
 LOH   
 Transcriptional repression • Loss of expression has been linked to tumor progression from DCIS to invasive carcinoma (39) 
 Posttranscriptional repression • When reexpressed, enhances growth inhibitory effect by promoting autophagy, apoptosis, and G2–M cell cycle arrest (75) 
 miRNA • DIRAS3 repression by JMJD2A promotes breast cancer progression (76) 
Pancreatic cancer Deletion • DIRAS3 expression is downregulated in pancreatic cancer (48%) compared with normal pancreatic tissue (18%), plays a role as tumor suppressor gene (77) 
 Hypermethylation • Reexpression of DIRAS3 in PDAC cell lines blocks cell cycle progression at G1 phase by inhibiting PI3K/AKT and NF-κB signaling pathways in addition to inhibiting ERK activity (33, 57) 
Thyroid cancer Deletion • A key early event observed in 85% of follicular thyroid cancers (23) 
 Hypermethylation • Advanced pathologic stage, poor differentiation degree, and worse prognosis, lower 3-year survival rates (78) 
Colon cancer Downregulation • Advanced tumor grade (79) 
 LOH   
 Promoter methylation • Reexpression of DIRAS3 inhibits cells invasion and adhesion through suppressing EMT via the suppression of the Wnt/β-catenin signaling pathway (80) 
Esophageal carcinoma Deletion • Activation of the Wnt pathway, decreased overall survival, increased invasive capacity, cell cycle progression and epithelial-mesenchymal transition (30) 
 Downregulation   
HCC Promoter hypermethylation Somatic mutation • Tumor growth, angiogenesis, impaired apoptosis, hepatocarcinogenesis (54) 
Lung cancer Hypermethylation • Aberrant methylation profile compared with normal tissue (81) 
 Downregulation • Impaired cyclin D1, MMP-1, and MMP-2 expression, deregulated cancer cell growth and proliferation, invasion, impaired apoptosis (82) 
Multiple myeloma Downregulation • Downregulation results in over-angiogenic phenotype (53) 
Osteosarcoma Downregulation • Development of osteosarcoma formation, promotes cell proliferation by enhancing PI3K/AKT signaling, attenuates apoptosis (83) 
 Hypermethylation • Zebularine, a demethylating agent repressing DNMT1, prevents methylation of DIRAS3 and increase its expression, inhibits the viability, and promotes apoptosis in osteosarcoma cells. (84) 
  • Reduced GAS5 lncRNA and increased miR-221.  
  • Overexpression of GAS5 suppresses the proliferation, migration, and EMT of osteosarcoma cells by enhancing DIRAS3 expression, suppresses tumor volume, Ki-67 and PCNA staining (85) 
Prostate cancer Downregulation • Overexpression inhibits cell proliferation, colony formation, invasion, and induces apoptosis (86) 
 Hypermethylation   
 Targeting of 3′UTR of DIRAS3 by miR221/222 • Lower DIRAS3 and higher miRNA–221/222 levels correlate with more aggressive prognosis, suggested as a biomarker for disease progression (4) 
Renal cancer Decreased mRNA levels • Advanced tumor stage, distant metastasis phenotype, lower survival rate (87) 
SCC Aberrant methylation • Development and progression of tongue SCC (88) 
  • Concurrent reexpression of p53 and DIRAS3 effectively induces cell death and decreases the tumor volume in HNSCC xenograft mouse model (89) 

Abbreviations: DCIS, ductal carcinoma in situ; EMT, epithelial–mesenchymal transition; HNSCC, squamous cell carcinoma of the head and neck; MMP, matrix metalloproteinase; SCC, squamous cell carcinoma.

A substantial amount of literature has grown over the last 2 decades regarding the function of DIRAS3 in tumor suppression, autophagy, and tumor dormancy, as well as its emerging role as a target for anticancer therapy. This review highlights this progress and places in perspective DIRAS3's role in human cancer.

DIRAS3 is an RAS-related GTPase with a distinctive NTE

The RAS superfamily is composed of more than 150 small GTPases divided into five major subfamilies based on the primary sequence of the structural motifs. DIRAS3 belongs to the RAS subfamily of RAS-related proteins (9). Despite encoding structurally similar proteins, the genomic structure of the RAS family of genes differs in length as well as coding sequence. Some RAS genes, such as KRAS, can extend over 40 kb, whereas DIRAS3 is contained in 8 kb, with one intron and two exons that encode a 26 kDa GTPase (10). DIRAS3 contains a unique extension of 34 amino acids at the N-terminus, which is rarely encountered in other RAS family members (Fig. 1A). Functional analysis has shown that the NTE is required for blocking malignant transformation by mutant KRAS and HRAS, disrupting RAS dimers and clusters, and inhibiting growth of normal and malignant cells, as well as inducing autophagy, suggesting that the NTE region plays an important role in DIRAS3 function (2, 11).

Figure 1.

Domain organization, amino acid sequence, and mechanism of regulation of DIRAS proteins. A, The imprinted DIRAS3 gene and its gene product. The domain organization of the DIRAS3 protein is shown and compared with the classical RAS family protein KRAS (KRAS4B). Both proteins have a similar domain structure: The GTP/GDP-binding domain (G-domain) that is composed of 5 conserved fingerprint motifs G1–G5, the hypervariable region (HVR), and CAAX motif for membrane association. DIRAS3 differs from KRAS by a unique NTE. B, Protein sequence alignment of the DIRAS 1, 2, and 3 with KRAS as reference. The length and amino acid composition of the NTE in DIRAS3 (34 aa), compared with DIRAS1&2 (4 aa). The putative myristoylation site G2 is highlighted in red. The specific deviations within the functional motifs (shaded in cyan), including the P-loop and the effector-binding switch I and II, are highlighted in bold and pink (see text for detailed explanation). The residues of polybasic clusters in the hypervariable region are in blue. The predicted prenyl cysteine in the CAAX motif (cysteine–aliphatic–aliphatic–any aa) is in bold. Multiple sequence alignment was performed at https://www.uniprot.org/ for KRAS (P01116–2), DIRAS1 (O95057), DIRAS2 (Q96HU8), and DIRAS3 (O95661). The boundary for each motif/region is based on the review paper by Nakhaei-Rad and collagues (90). C, Loss of DIRAS3 function in cancers results from genetic, epigenetic, transcriptional, and posttranscriptional changes. Hypermethylation of CpG islands in the promoter regions of both DIRAS3 alleles downregulates DIRAS3 expression in 20% of cases. LOH at the DIRAS3 locus on chromosome 1p31 has been found in 30% to 40% of ovarian and breast cancers. The A2 segment of the DIRAS3 promoter (bp −409 to −385) contains an E2F-binding site. Binding of both E2F1 and E2F4 inhibits DIRAS3 promoter activity. Transcriptional repression by E2F1 and E2F4 and their complexes with histone deacetylase (HDAC) has been demonstrated in 20% to 30% in breast and ovarian cancers. Also, at the DIRAS3 locus, GNG12-AS1 lncRNA, covers a 370 kb genomic region that starts near the GNG12 promoter and runs through DIRAS3 in an antisense orientation. Transcriptional silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating transcriptional interference. DIRAS3 mRNA in normal cells exhibits half lives of 17 to 19 hours, DIRAS3 mRNA in ovarian cancer cell lines has a significantly reduced half-life of 8 to 9 hours. Thus, the half-life of the DIRAS3 mRNA is nearly 2.5 times shorter than that in normal ovarian epithelial cells. The shorter half-life relates to the stability of DIRAS3 mRNA which is regulated by binding of DIRAS3 mRNA to HuR-ARE proteins through U- or AU-rich sequences in the 3′-UTR of DIRAS3 mRNA. Binding stabilizes mRNA and enhances translation by shuttling between the nucleus and the cytoplasm. Decreased HuR-ARE binding activity reduces its ability to stabilize the DIRAS3 mRNA and contributes to faster mRNA turnover in cancer cells.

Figure 1.

Domain organization, amino acid sequence, and mechanism of regulation of DIRAS proteins. A, The imprinted DIRAS3 gene and its gene product. The domain organization of the DIRAS3 protein is shown and compared with the classical RAS family protein KRAS (KRAS4B). Both proteins have a similar domain structure: The GTP/GDP-binding domain (G-domain) that is composed of 5 conserved fingerprint motifs G1–G5, the hypervariable region (HVR), and CAAX motif for membrane association. DIRAS3 differs from KRAS by a unique NTE. B, Protein sequence alignment of the DIRAS 1, 2, and 3 with KRAS as reference. The length and amino acid composition of the NTE in DIRAS3 (34 aa), compared with DIRAS1&2 (4 aa). The putative myristoylation site G2 is highlighted in red. The specific deviations within the functional motifs (shaded in cyan), including the P-loop and the effector-binding switch I and II, are highlighted in bold and pink (see text for detailed explanation). The residues of polybasic clusters in the hypervariable region are in blue. The predicted prenyl cysteine in the CAAX motif (cysteine–aliphatic–aliphatic–any aa) is in bold. Multiple sequence alignment was performed at https://www.uniprot.org/ for KRAS (P01116–2), DIRAS1 (O95057), DIRAS2 (Q96HU8), and DIRAS3 (O95661). The boundary for each motif/region is based on the review paper by Nakhaei-Rad and collagues (90). C, Loss of DIRAS3 function in cancers results from genetic, epigenetic, transcriptional, and posttranscriptional changes. Hypermethylation of CpG islands in the promoter regions of both DIRAS3 alleles downregulates DIRAS3 expression in 20% of cases. LOH at the DIRAS3 locus on chromosome 1p31 has been found in 30% to 40% of ovarian and breast cancers. The A2 segment of the DIRAS3 promoter (bp −409 to −385) contains an E2F-binding site. Binding of both E2F1 and E2F4 inhibits DIRAS3 promoter activity. Transcriptional repression by E2F1 and E2F4 and their complexes with histone deacetylase (HDAC) has been demonstrated in 20% to 30% in breast and ovarian cancers. Also, at the DIRAS3 locus, GNG12-AS1 lncRNA, covers a 370 kb genomic region that starts near the GNG12 promoter and runs through DIRAS3 in an antisense orientation. Transcriptional silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating transcriptional interference. DIRAS3 mRNA in normal cells exhibits half lives of 17 to 19 hours, DIRAS3 mRNA in ovarian cancer cell lines has a significantly reduced half-life of 8 to 9 hours. Thus, the half-life of the DIRAS3 mRNA is nearly 2.5 times shorter than that in normal ovarian epithelial cells. The shorter half-life relates to the stability of DIRAS3 mRNA which is regulated by binding of DIRAS3 mRNA to HuR-ARE proteins through U- or AU-rich sequences in the 3′-UTR of DIRAS3 mRNA. Binding stabilizes mRNA and enhances translation by shuttling between the nucleus and the cytoplasm. Decreased HuR-ARE binding activity reduces its ability to stabilize the DIRAS3 mRNA and contributes to faster mRNA turnover in cancer cells.

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DIRAS3 requires membrane association for its function

Like other members of the RAS family, DIRAS3 has a C-terminal prenylation site that mediates attachment of the protein to the inner leaflet of the cell surface membrane. Mutation at C226 or deletion of the C-terminus prevents membrane localization of DIRAS3 and reduces its biological function, supporting the concept that its function depends on posttranslational prenylation at C226 and its association with the plasma membrane (2, 12). Recently, our group discovered that the DIRAS3 NTE contains a membrane-targeting motif and an N-myristoylation site (13). We speculate that NTE and CAAX motif can stabilize DIRAS3 on the membrane by double anchoring and facilitating the known regulation of KRAS by DIRAS3.

DIRAS3 has been lost from the murine genome during evolution

The RAS super family of small GTPases is found in the genome of many organisms, including eukaryotes as well as prokaryotes (14). Many mammalian species express RAS super family genes including DIRAS3 and different members of this family appear to have emerged very early during evolution (15). DIRAS3 has been lost from the murine genome when lineages diverged. Consequently, DIRAS3 is found in humans, primates, cows, and pigs, but not in mice or rats. The deletion in rodents of this otherwise much-conserved gene appears to relate to the hypothetical location of DIRAS3 at the distal end of a murine chromosome. The telomeric flank of the human DIRAS3 gene is syntenic with regions that are adjacent to the telomere of mouse chromosome 3, consistent with deletion of DIRAS3 during telomere formation (16). The absence of DIRAS3 from the murine genome has prevented traditional studies of tumor suppression in genetically engineered mice and suggests that murine models may not be optimal for studies of processes that can depend upon DIRAS3 including RAS regulation, autophagy, and dormancy. Mice do express DIRAS1 and DIRAS2, which appear to participate in many DIRAS3-mediated functions, including autophagy.

DIRAS1 and DIRAS2 share several functions with DIRAS3, despite truncated N-terminal domains and alterations in GTPase-mediating residues. The DIRAS family proteins (DIRAS1, 2, and 3) share 50% to 60% amino acid sequence homology (Fig. 1B). Like DIRAS3, DIRAS1 and DIRAS2 are downregulated in human ovarian cancer, associated with a poor prognosis (17). Reexpression of DIRAS1 and DIRAS2 induce autophagy and suppress the growth of human and murine ovarian cancers (17). DIRAS1 and DIRAS2 differ primarily from DIRAS3 in the truncation of the 34-amino acid NTE of DIRAS3 to 4 amino acids in DIRAS1 and DIRAS2. All three molecules share a conserved cysteine prenyl binding site in the C-terminal CAAX motif. Consequently, all three proteins can associate with the inner leaflet of the cell-surface membrane, but myristoylation and binding of the N-terminus to the plasma membrane is a distinctive feature of DIRAS3. Most RAS GTPases serve as molecular switches that are “on” when GTP is bound, promoting signal transduction, and “off” after GTP is cleaved and GDP remains bound to the RAS protein, preventing signal transduction. A nucleotide exchange factor is required to remove GDP and permit binding of fresh GTP. Compared with KRAS, all three DIRAS proteins share a conserved GTP-binding domain with identical G4 and G5 motif sequences. Although GTP binds to DIRAS3 with high affinity, the protein has low intrinsic GTPase activity and is likely in a constitutively activated GTP-bound state (2). Similarly, more than half of DIRAS1 and DIRAS2 proteins are found in their GTP-bound forms within cells (18). Residues A59 and Q61 in the G3/Switch II region of KRAS are known to be critical for GTP hydrolysis. Consistent with decreased GTPase activity, these positions are changed to T63 and S65 in DIRAS1 and DIRAS2, and to K93 and G95 in DIRAS3. (19). While a conserved glycine (G60 in KRAS), which is important for nucleotide exchange, is unchanged in DIRAS1&2 (G64), it is altered to S94 in DIRAS3. Finally, a majority of RAS family members contain a glycine at position 12 in the G1/phosphate-binding loop (P-loop) which provides one of the most common sites for KRAS mutational activation (19, 20). Although this residue is conserved in DIRAS1 and DIRAS2, it is replaced by alanine in DIRAS3 (A46), consistent with the possibility that DIRAS3 does not act as a molecular switch (2), but rather as a consistent inhibitor of RAS function. Crystal structures of DIRAS1 (PDB ID: 2GF0) and DIRAS2 (PDB ID: 2ERX) proteins have been solved, but the structure of full-length DIRAS3 has not yet been established. Recent studies unraveling the crystal structure of a DIRAS2/3 chimera (PDB ID: 6NAZ) provided structural insight into the fold of the switch II region of DIRAS3. This is important for interaction with Beclin1 protein (21), which has a central role in mammalian autophagy.

DIRAS3 is maternally imprinted and expressed only from the paternal allele

Genomic imprinting, a mechanism of epigenetic regulation that allows a gene to be expressed at half dosage in a parent-of-origin–specific manner, is involved in normal growth and development (22). A maternally imprinted gene, DIRAS3, has three differentially methylated CpG regions (DMRs) in the maternal allele, corresponding to the DIRAS3 upstream CpG island (CpG I), the transcription start site (CpG II), and exon 2 of the DIRAS3 promoter (CpG III). In cancers tested to date, no mutations of DIRAS3 have been detected in the coding and promoter regions (23, 24). As every DMR of the maternal DIRAS3 allele is hypermethylated and silenced in normal cells, monoallelic expression can occur only from the paternal allele (25). In cancer cells, expression from the paternal copy of DIRAS3 can be lost by different mechanisms, resulting in loss of function in a single event (Fig. 1C), analogous to loss of function in a “single step” with inactivating germ line mutations of other tumor suppressor genes.

DIRAS3 expression can be downregulated in cancer by genetic and epigenetic mechanisms

Loss of DIRAS3 function in cancer has been shown to result from genetic, epigenetic, transcriptional, and posttranscriptional mechanisms. LOH at the DIRAS3 locus on chromosome 1p31 has been found in 30% to 40% of ovarian and breast cancers (24). Uniparental disomy (UPD) of DIRAS3 is a mechanism of LOH where both chromosome segments containing the DIRAS3 gene loci are inherited from the mother due to parental segregation errors (26). Here, a tumor suppressor gene will not be expressed, and the individual is put at a greater risk for several cancers. UPD is often a result of a germline event. However, this event can also occur in a portion of somatic cells as a result of mitotic cell division error and it is called “acquired UPD.” Acquired UPD is a common phenomenon in a variety of hematologic and solid tumors, and is reported to constitute 20% to 80% of the LOH seen in human tumors (27). Hypermethylation of CpG islands in the promoter regions of both DIRAS3 alleles downregulates DIRAS3 expression in 20% of cases (10).

DIRAS3 expression can be decreased by transcriptional regulation

Specific transcriptional repressors can contribute to decreased DIRAS3 expression. The A2 segment of the DIRAS3 promoter (bp −409 to −385) contains an E2F-binding site. The binding of both E2F1 and E2F4 inhibits DIRAS3 promoter activity (7). Transcriptional repression by E2F1 and E2F4 and their complexes with histone deacetylase (HDAC) has been demonstrated in 20% to 30% of breast and ovarian cancers (7).

Posttranscriptional mechanisms have been implicated in DIRAS3 downregulation

Posttranscriptional gene regulation also plays an important role in regulating the efficiency of translation and stability of mRNA. The equilibrium between the synthesis of mRNA and its degradation determines the steady-state levels of individual mRNAs. Normal ovarian epithelial cells have much higher steady-state levels of DIRAS3 mRNA than ovarian cancer cells do. Whereas DIRAS3 mRNA in normal cells exhibits a half-life of 17 to 19 hours, DIRAS3 mRNA in ovarian cancer cell lines has a significantly reduced half-life of 8 to 9 hours (8). Thus, the half-life of the DIRAS3 mRNA is nearly 2.5 times shorter than that in normal ovarian epithelial cells. The shorter half-life relates to the stability of DIRAS3 mRNA which is regulated by binding of HuR-ARE proteins to DIRAS3 mRNA through U- or AU-rich sequences in the 3′-untranslated region (UTR) of DIRAS3 mRNA. Binding stabilizes mRNA and enhances translation by shuttling between the nucleus and the cytoplasm (3). Decreased HuR-ARE binding activity in ovarian cancer reduces its ability to stabilize the DIRAS3 mRNA and contributes to faster mRNA turnover in ovarian cancer cells (3).

Another important posttranscriptional downregulation of DIRAS3 is due to long noncoding RNAs (lncRNA) that can regulate gene expression by inhibiting specific translation or inducing cleavage of target mRNAs. Through bioinformatics analysis, Chen and colleagues predicted two microRNAs (miR-221 and miR-222) that might target the 3′UTR region of DIRAS3 mRNA and confirmed the direct interaction of miR-221 or -222 with a target site on the 3′UTR of DIRAS3 (4). This study demonstrates for the first time that prostate cancer cells have decreased levels of DIRAS3 due to inhibited translation and induced cleavage of DIRAS3 mRNAs after interacting with miR-221 or -222. Imprinted gene clusters are also regulated by lncRNAs. DIRAS3 is located within an intron of a long noncoding RNA named LOC100289178 or GNG12-AS1. At the DIRAS3 locus, GNG12-AS1 lncRNA covers a 370-kb genomic region that starts near the GNG12 promoter and runs through DIRAS3 in an antisense orientation. In normal cells, GNG12-AS1 is expressed from both maternal and paternal alleles. In cancer cell lines, however, GNG12-AS1 expression is decreased on the paternal allele, and this lncRNA is predominantly expressed from the maternal allele (25). Transcriptional silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating transcriptional interference. This upregulation of DIRAS3 expression is sufficient to impair cell-cycle progression (6).

Downregulation of DIRAS3 in a large fraction of cancers from multiple sites, argues for its importance as a tumor suppressor gene. With an imprinted gene like DIRAS3, downregulation in a single step can be biologically equivalent to the “second hit” in Knudson hypothesis for loss of tumor suppressor activity (28). Regulation of DIRAS3 may, however, be more complex. Some breast cancer cell lines lose their maternal-specific methylation of CpG island II, a phenomenon termed loss of imprinting (LOI; ref. 5). Consequently, imprinted genes in UPD regions might have distinct allele-specific gene-expression patterns (5, 8). The mechanism and importance of LOI in DIRAS3 CpG island II are still unknown.

DIRAS3 blocks mutant KRAS- and HRAS-mediated malignant transformation

As DIRAS3 has been lost from the murine genome, traditional knockout studies cannot be performed to document its tumor-suppressor function. Using a traditional cell culture model, coexpression of DIRAS3 has, however, been shown to block the transformation of murine NIH3T3 fibroblasts by constitutively active G12V KRAS or HRAS (12). Inhibition of RAS transformation requires the N-terminal domain of DIRAS3 and membrane association through the DIRAS3 C-terminal. Similar results were obtained with partially transformed MCF10a breast epithelial cells where KRAS- or HRAS G12V–induced anchorage-independent clonogenic growth was suppressed by DIRAS3. Overall, reexpression of DIRAS3 inhibits the growth of fully transformed cells from RAS-driven cancers, including pancreatic, lung, colon, and ovarian cancers (12). DIRAS3 can, however, also inhibit growth of cancers not driven by mutant RAS (29–31).

DIRAS3 blocks RAS function and signaling by disrupting RAS dimers and multimers

Mutant RAS-driven malignant progression contributes to approximately one third of human cancers. Regulation of RAS by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) has been well studied (32), as has activation of RAS by mutation (33), but other mechanisms that regulate RAS-driven signaling have received less attention. RAS dimerization, multimerization, or clustering is strongly associated with RAS signal activation (34). The specific mechanisms by which RAS activity is regulated have not been fully clarified and, apart from wtRAS itself, no physiologic oncogenic RAS clustering inhibitors have been identified (35). Recently, Sutton and colleagues found that DIRAS3 binds directly with RAS through its A5 helical domain to form DIRAS3-RAS heteromers and disrupts RAS dimerization and clustering, inhibiting RAF kinase and downstream MAPK signaling (Fig. 2; ref. 12). Disruption of KRAS cluster formation, like inhibition of RAS transformation, requires the N-terminus of DIRAS3 and interaction of DIRAS3 with the plasma membrane. Thus, DIRAS3 regulates oncogenic RAS activity by inhibiting RAS clustering and multimerization (12).

Figure 2.

DIRAS3 blocks RAS function and signaling by disrupting RAS dimers and multimers. RAS dimerization, multimerization, or clustering is strongly associated with RAS signal activation. DIRAS3 binds directly with RAS through its A5 helical domain to form DIRAS3-RAS heteromers and disrupts RAS dimerization and clustering, inhibiting RAF kinase and downstream MAPK signaling. Disruption of KRAS cluster formation, like inhibition of RAS transformation, requires the N-terminus of DIRAS3 and interaction of DIRAS3 with the plasma membrane. Thus, DIRAS3 regulates oncogenic RAS activity by inhibiting RAS clustering and multimerization.

Figure 2.

DIRAS3 blocks RAS function and signaling by disrupting RAS dimers and multimers. RAS dimerization, multimerization, or clustering is strongly associated with RAS signal activation. DIRAS3 binds directly with RAS through its A5 helical domain to form DIRAS3-RAS heteromers and disrupts RAS dimerization and clustering, inhibiting RAF kinase and downstream MAPK signaling. Disruption of KRAS cluster formation, like inhibition of RAS transformation, requires the N-terminus of DIRAS3 and interaction of DIRAS3 with the plasma membrane. Thus, DIRAS3 regulates oncogenic RAS activity by inhibiting RAS clustering and multimerization.

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DIRAS3 can form a complex with HRAS and c-RAF inhibiting c-RAF activity

In addition to the role of DIRAS3 in disrupting KRAS clustering, DIRAS3 can also interfere with HRAS signal transduction through c-RAF to MEK. In normal signaling, activated HRAS dimers bind b-RAF and c-RAF, permitting b-RAF-c-RAF heterodimerization. In the presence of DIRAS3, HRAS is still able to bind its effector c-RAF protein (36), but the multimeric complex consists of DIRAS3, c-RAF, and active HRAS is more stable than the two protein complexes HRAS-c-RAF or HRAS-DIRAS3 respectively. The consequence of this complex formation is DIRAS3-mediated recruitment and anchorage of c-RAF to components of the membrane skeleton, suppression of c-RAF/b-RAF heterodimerization, and inhibition of c-RAF kinase activity.

DIRAS3 reexpression downregulates PI3K/AKT, and JAK/STAT, as well as RAS/ERK signaling

DIRAS3 functions as a tumor suppressor through its inhibitory effects on the PI3K/AKT, RAS/ERK, and JAK/STAT signaling pathways. Inducible expression of DIRAS3 at physiologic levels in different ovarian cancer cell lines reduces epidermal growth factor (EGF)–stimulated PI3K activity, resulting in a reduction of phosphatidylinositol 3,4,5-trisphosphate (PIP3) production, decreased membrane localization, and reduced phosphorylation of AKT (37). DIRAS3 reexpression also reduces phosphorylated ERK levels. As the key upstream regulator of ERK activation is RAS, RAS-GTP pull-down experiments detected decreased levels of GTP-bound activated RAS and decreased membrane localization of RAS after DIRAS3 reexpression. The significance of dual pathway (PI3K and RAS-MAPK) inhibition by DIRAS family members including DIRAS1, DIRAS2, and DIRAS3 is underlined by their ability to regulate nuclear localization of major transcription factors such as FOXO3a and TFEB in maintaining cellular homeostasis (17). In addition to PI3K and ERK, DIRAS3 reexpression can also interrupt JAK/STAT signaling by binding directly to STAT3 and preventing its translocation to focal adhesion complexes and the nucleus (38).

Reexpression of DIRAS3 inhibits cycle progression in malignant cells

Growth suppressing actions of DIRAS3 correlate with inhibition of cell-cycle progression, although DIRAS3 can also kill malignant cells through mechanisms described below. DIRAS3 downregulates cyclin D1 promoter activity and increases p21WAF1 levels in breast and ovarian carcinomas shown in cell culture studies as well as histopathologic data and clinical outcomes (1, 31, 39). Further efforts to identify its effects on cell-cycle regulation mechanisms indicate that DIRAS3 blocks cell-cycle progression at the G1 phase through modulation of several key G1 regulatory proteins, such as p21WAF1, p27kip1, CDK2, CDK4, and cyclins A and D1 in pancreatic and breast cancer cells (40, 41). The activation of the CDK4-cyclin D1 complex is crucial for the transition from early to mid-G1 phase. Transition from mid-G1 to S phase is regulated by activation of the CDK2-cyclin E complex. Progression through late G1 to S phase also requires the presence of the CDK2-cyclin A complex. When reexpressed, DIRAS3 increases the expression of the cyclin-dependent kinase inhibitors p21WAF1 and p27Kip1 through the inhibition of PI3K/AKT and RAS/ERK signaling (Fig. 3). Increased expression of p21WAF1 and p27Kip1 prevent CDK2 and CDK4 from stimulating cell cycle to progress from early to mid-G1 and from G1 to S phase while DIRAS3 also decreases cyclin A and cyclin D1 levels propagating a cell cycle arrest. Furthermore, an additional mechanism by which DIRAS3 regulates cell cycle is inhibition of the JAK/STAT3 signaling pathway by directly reducing STAT3 phosphorylation (Tyr705) thereby decreasing cyclin D1 accumulation which results in S phase arrest (42). Moreover, transcriptional silencing of GNG12-AS1, a long noncoding RNA that runs through DIRAS3 in an antisense orientation, causes concomitant upregulation of DIRAS3, reported to impair cell-cycle progression from G1 to S (6). These observations collectively establish the role of DIRAS3 as a negative regulator of cell growth, through interaction with master regulators of the cell cycle.

Figure 3.

Reexpression of DIRAS3 inhibits cycle progression in malignant cells. DIRAS3 downregulates cyclin D1 promoter activity and increases p21WAF1 levels and blocks cell-cycle progression at the G1 phase through modulation of several key G1 regulatory proteins, such as p21WAF1, p27kip1, CDK2, CDK4, and cyclins A and D1. When reexpressed, DIRAS3 increases the expression of the cyclin-dependent kinase inhibitors p21WAF1 and p27Kip1 through the inhibition of PI3K/AKT and RAS/ERK signaling. Increased expression of p21WAF1 and p27Kip1 prevent CDK2 and CDK4 from stimulating cell cycle to progress from early to mid-G1 and from G1 to S phase while DIRAS3 also decreases cyclin A and cyclin D1 levels propagating a cell cycle arrest. Furthermore, an additional mechanism by which DIRAS3 regulates cell cycle is inhibition of JAK/STAT3 signaling pathway by directly reducing STAT3 phosphorylation (Tyr705) thereby decreasing cyclin D1 accumulation which results in S phase arrest.

Figure 3.

Reexpression of DIRAS3 inhibits cycle progression in malignant cells. DIRAS3 downregulates cyclin D1 promoter activity and increases p21WAF1 levels and blocks cell-cycle progression at the G1 phase through modulation of several key G1 regulatory proteins, such as p21WAF1, p27kip1, CDK2, CDK4, and cyclins A and D1. When reexpressed, DIRAS3 increases the expression of the cyclin-dependent kinase inhibitors p21WAF1 and p27Kip1 through the inhibition of PI3K/AKT and RAS/ERK signaling. Increased expression of p21WAF1 and p27Kip1 prevent CDK2 and CDK4 from stimulating cell cycle to progress from early to mid-G1 and from G1 to S phase while DIRAS3 also decreases cyclin A and cyclin D1 levels propagating a cell cycle arrest. Furthermore, an additional mechanism by which DIRAS3 regulates cell cycle is inhibition of JAK/STAT3 signaling pathway by directly reducing STAT3 phosphorylation (Tyr705) thereby decreasing cyclin D1 accumulation which results in S phase arrest.

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DIRAS3 inhibits cancer cell migration and metastasis

Metastatic tumor progression is a multistep process that begins with the local invasion of the primary tumor into the surrounding tissue, accompanied by the spreading of cancer cells through lymphatics and blood vessels, producing metastases at distant locations. Since active tumor-cell migration is required for tumor-cell invasion and metastases, a primary goal of tumor biology has been to elucidate factors that control the migratory behavior of such cells. A meta-analysis including more than 1,200 metastatic or recurrent breast cancers, identified DIRAS3 as a potential metastasis suppressor gene where allelic loss and methylation of DIRAS3 and the 1p31 chromosomal region correlated with increased risk of metastasis (43).

DIRAS3 has been shown to inhibit signaling through the Stat3 and FAK/Rho pathways. FAK activity is triggered by a variety of growth factors, as well as the binding of receptors and integrins. Phosphorylation of FAK spreads signals to a variety of downstream effectors to control disassembly and reaggregation of actin microfilaments needed for active cell movement. DIRAS3 inhibits the phosphorylation of FAK which prevents Rho-mediated paxillin and vinculin phosphorylation, and subsequent actin stress-fiber formation (38). DIRAS3 and Stat3 interactions are exceptionally compelling in that Stat3 signaling are vital for both cell growth and motility. The majority of ovarian cancers not only secrete IL6, but also express the IL6 receptor, which mediates the phosphorylation of Stat3 by JAK. Phospho-STAT3 translocates to the nucleus to stimulate the transcription of certain genes, including N-cadherin and vimentin (44). DIRAS3 binds to and sequesters Stat3 in the cytoplasm, preventing translocation of Stat3 to the nucleus and focal adhesion complexes. The increased arrest of Stat3 in the cytoplasm prevents transcription of adhesion molecules such as N-cadherin and vimentin and prevents both proliferation and motility by inhibiting a single critical mediator (Fig. 4). Negative regulation of RAS/MEK/ERK signaling by DIRAS3 further supports this model as both Stat3 and FAK are downstream effectors of ERK. Therefore, inhibition of cell migration is an indirect physiologic consequence of DIRAS3-mediated ERK inhibition (11). Autophagy has been proposed to play an important role in regulating tumor invasion (45). Recent studies revealed that IL6 inhibits the formation of autophagosomes at the migratory front, which is associated with a faster migration of cells. If this is the case, DIRAS3-mediated stimulation of autophagy, as described below, might also inhibit cell migration (46).

Figure 4.

DIRAS3 inhibits cancer cell migration and metastasis through the Stat3 and FAK/Rho pathways. DIRAS3 inhibits the phosphorylation of FAK which prevents Rho-mediated paxillin and vinculin phosphorylation, and the subsequent actin stress fiber formation. DIRAS3 and Stat3 interaction is exceptionally compelling in that Stat3 signaling is vital for both cell growth and motility. DIRAS3 binds to and sequesters Stat3 in the cytoplasm, preventing translocation of Stat3 to the nucleus and focal adhesion complexes. Increased arrest of Stat3 in the cytoplasm prevents transcription of adhesion molecules such as N-cadherin and vimentin and prevents both proliferation and motility by inhibiting a single critical mediator.

Figure 4.

DIRAS3 inhibits cancer cell migration and metastasis through the Stat3 and FAK/Rho pathways. DIRAS3 inhibits the phosphorylation of FAK which prevents Rho-mediated paxillin and vinculin phosphorylation, and the subsequent actin stress fiber formation. DIRAS3 and Stat3 interaction is exceptionally compelling in that Stat3 signaling is vital for both cell growth and motility. DIRAS3 binds to and sequesters Stat3 in the cytoplasm, preventing translocation of Stat3 to the nucleus and focal adhesion complexes. Increased arrest of Stat3 in the cytoplasm prevents transcription of adhesion molecules such as N-cadherin and vimentin and prevents both proliferation and motility by inhibiting a single critical mediator.

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DIRAS3 induces autophagy

In normal cells, autophagy is a process by which cells catabolize amino and fatty acids from aging organelles and long-lived proteins to maintain homeostasis, ensure quality control of cell contents, and supply energy (47). In malignant cells, autophagy can play a dual role by producing energy to enhance cancer cell survival in nutrient-poor environments, but also by inducing type 2 autophagic cancer cell death (48). DIRAS3 is essential for the induction of autophagy by nutrient and amino acid starvation or by treatment with rapamycin in normal and malignant human cells (40, 43). Knockdown of DIRAS3 with siRNA impairs the induction of autophagy in multiple ovarian cancer cell lines and cultures of normal ovarian epithelial cells (49, 50). DIRAS3 reexpression can upregulate autophagy at four levels: (i) blocking PI3K/AKT signaling, downregulating mTOR, and therefore decreasing inhibitory phosphorylation of ULK1 to induce autophagy, (ii) displacing Bcl-2 from Beclin1 to form the autophagy initiation complex, (iii) inhibiting RAS/MAPK and PI3K, maintaining FOXO3a in the nucleus to induce critical gene expressions that participate in autophagy (e.g., LC3, Atg4), and (iv) facilitating fusion of autophagosomes and lysosomes through Rab7 (37, 49, 50). It also colocalizes with LC3 in the autophagosome membrane to play a critical role in autophagosome formation (Fig. 5). According to a study conducted in glioblastoma, overexpression of DIRAS3 decreases the activity of RAS in glioma cells, indirectly suppressing mTOR activity and ultimately initiating autophagy and inhibiting proliferation (51). DIRAS3-induced autophagy is accompanied by several distinct features such as increased numbers of autophagic vacuoles in the cytoplasm, membrane-associated forms of LC3‐I and LC3‐II, and typical ultrastructural changes. These effects are only associated with full length DIRAS3, but not N-terminal deleted (NTD) mutant DIRAS3, supporting the concept that the N‐terminal extension plays an important role in its function. Interestingly, murine cells still undergo autophagy in the absence of DIRAS3. As noted above, this may be explained by the presence of two other homologous RAS-related GTPases, DIRAS1 and DIRAS2, which share 50% to 60% homology with DIRAS3 (17).

Figure 5.

DIRAS3 is essential for the induction of autophagy. DIRAS3 can regulate autophagy at four levels: (i) blocking PI3K/AKT signaling, downregulating mTOR and therefore decreasing inhibitory phosphorylation of ULK1 to induce autophagy, (ii) displacing Bcl-2 from Beclin1 to form the autophagy initiation complex, (iii) inhibiting RAS/MAPK and PI3K, maintaining FOXO3a in the nucleus to induce critical gene expressions that participate in autophagy (LC3, Atg4), and (iv) facilitating fusion of autophagosomes and lysosomes through Rab7. It also colocalizes with LC3 in the autophagosome membrane to play a critical role in autophagosome formation.

Figure 5.

DIRAS3 is essential for the induction of autophagy. DIRAS3 can regulate autophagy at four levels: (i) blocking PI3K/AKT signaling, downregulating mTOR and therefore decreasing inhibitory phosphorylation of ULK1 to induce autophagy, (ii) displacing Bcl-2 from Beclin1 to form the autophagy initiation complex, (iii) inhibiting RAS/MAPK and PI3K, maintaining FOXO3a in the nucleus to induce critical gene expressions that participate in autophagy (LC3, Atg4), and (iv) facilitating fusion of autophagosomes and lysosomes through Rab7. It also colocalizes with LC3 in the autophagosome membrane to play a critical role in autophagosome formation.

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DIRAS3 induces autophagic cancer cell death

Overexpression of DIRAS3 at supraphysiologic levels inhibits the growth of ovarian and breast cancer cells and xenografts by inducing caspase‐independent, calpain‐dependent apoptosis (29). By contrast, restoring DIRAS3 expression at physiologic levels is associated with minimal apoptosis, but with substantial necroptosis, involving both necrosis and activation of the RIPK1-RIPK3 complex (52). Growth inhibition by DIRAS3 is blocked in ovarian cancer cells by knockdown of ATG5, consistent with type 2 autophagic cell death.

DIRAS3 inhibits angiogenesis

Angiogenesis is a process required in normal reproduction and wound healing where neovascularization is tightly regulated. Unregulated angiogenesis, however, can lead to several angiogenic diseases and is essential for the progression of solid tumor growth and metastasis. For metastatic cancer cells, the angiogenic network supplies nutrients, oxygen, and immune cells, while eliminating metabolic waste products. In the absence of vascular support, tumors may become necrotic or even apoptotic. The tumor microvasculature is derived from normal endothelium and pericytes but exhibits distinctive characteristics. Ria and colleagues analyzed bone marrow angiogenesis in multiple myeloma and found that DIRAS3 was 4.3 times less expressed in multiple myeloma endothelial cells than in normal endothelial cells (53). Further studies on another highly vascular tumor, hepatocellular carcinoma (HCC), indicated that overexpression of DIRAS3 in the Hep3B xenograft model was associated with a significant decrease in CD31-positive microvessels accompanied by a decrease in mTOR activity and diminished VEGF and HIF1a expression (54). These data suggest that the antiangiogenic effects of DIRAS3 are likely mediated by mTOR signaling interfering with the HIF1a-mediated VEGF signaling pathway.

DIRAS3 induces and maintains tumor dormancy

DIRAS3 reexpression results in autophagic ovarian cancer cell death in culture within 72 hours (50). Reexpression of DIRAS3 from a doxycycline inducible promoter establishes dormancy in xenografts in immunosuppressed nu/nu mice, where cancer cells remain viable for weeks after induction of DIRAS3. As soon as DIRAS3 levels are reduced in dormant cells by removing doxycycline, xenografts grow promptly to kill the host (55). Dormant ovarian cancer cells require autophagy to survive in a nutrient-poor environment. Inhibition of autophagy with chloroquine while cancer cells are dormant dramatically delays and reduces outgrowth of xenografts when DIRAS3 levels are reduced, indicating that autophagy contributes to the survival of dormant cells. Further studies with DIRAS3-inducible ovarian cancer cells showed that viability can be retained if cell culture media was supplemented with VEGF, IL8 and insulin-like growth factor (IGF) which rescue cancer cells from DIRAS3-mediated autophagic death. Interestingly, multiple antiangiogenic factors such as TIMP3, TSP1, Ang1, Ang2, Ang4, and CDH1 are upregulated in dormant cells and downregulated during the transition to recurrence (56).

The clinical relevance of the DIRAS3-inducible xenograft model is supported by the observation that dormant, drug-resistant ovarian cancer cells found in scars on the peritoneal surface at “second look” operations after primary surgery and chemotherapy express DIRAS3 and autophagy-related proteins in 80% of cases, compared with 20% in primary cancers (49). Although a subset of DIRAS3 expressing cancer cells might be selected by chemotherapy, preliminary data argue against this possibility. Sutton and colleagues recently demonstrated that amino acid withdrawal downregulates mTOR which in turn decreases binding of E2F1/4 to the DIRAS3 promoter, upregulates DIRAS3, and induces autophagy (57). This supports the view that malignant cells can transcriptionally upregulate DIRAS3 under nutrient-poor conditions found in poorly vascularized scars on the peritoneal cavity, inducing autophagy that could sustain dormant, drug-resistant cancer cells.

DIRAS3 inhibits adipogenesis

A variety of other functions of DIRAS3 in nonmalignant conditions have been reported. DIRAS3 has been identified as a negative regulator of adipogenesis and an activator of autophagy in adipose-derived stromal/progenitor cells (ASCs) possibly related to inhibition of AKT–mTOR signaling (58). Under proadipogenic conditions, DIRAS3 strongly counteracts insulin and IGF-1 activity and reduces adipogenic differentiation products such as FABP4, perilipin, and adiponectin in immature adipocytes. DIRAS3 appears to be involved in the reduced adipogenic capacity in subcutaneous white adipose tissue of formerly obese donors after long-term weight loss. DIRAS3 knockout models accelerate premature senescence in human white adipose stromal/progenitor cells where an increased number of senescent ASCs are observed (59). This diverse function of DIRAS3 disables cellular senescence and induces lifespan extension in animal models by reducing AKT-mTOR signaling.

DIRAS3 promotes endometriosis

DIRAS3 expression has also been studied in ectopic and eutopic endometrium of women with endometriosis. Ectopic endometrium highly expressed DIRAS3 on mRNA and protein levels compared with eutopic and normal endometrium (P < 0.005; ref. 60). DIRAS3 was expressed in normal endometrium and upregulated in ectopic endometrium, whereas expression was lost during malignant transformation, suggesting a role of DIRAS3 in infertility and tumorigenesis of endometriosis (60).

DIRAS3 is a potential biomarker for response to autophagy inhibitors

Based on the fact that DIRAS3 is a critical component of the autophagic machinery, the expression of DIRAS3 could mark cells that are susceptible to inhibition of autophagy. Antiautophagic therapy has been evaluated in the clinic, often in patients with large volumes of advanced metastatic cancer and without considering whether a large fraction of cancer cells is, in fact, undergoing autophagy (65). Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are FDA-approved drugs with the ability to inhibit autophagy functionally. Recent studies suggest that CQ primarily inhibits autophagy by impairing autophagosome fusion with lysosomes or impairment of Golgi and endosomal functions which might contribute to impairment of fusion, rather than by affecting the acidity and/or degradative activity of this organelle (66). Our previous studies showed that treatment of dormant DIRAS3-expressing human ovarian cancer cells with the autophagy inhibitor CQ delays the outgrowth of dormant ovarian cancer cells after downregulating DIRAS3 expression in this xenograft model (50) that it mimics observations in ovarian cancer patients with positive second-look operations where small nodules of persistent DIRAS3 expressing, drug-resistant ovarian cancer cells are undergoing autophagy judged by punctate LC3 in more than 80% of cases. Aside from ovarian carcinomas, this strategy has been shown to be effective in glioblastoma cells where DIRAS3 reexpression combined with blockade of autophagic flux by CQ enhanced the cytotoxicity of DIRAS3, caused accumulation of autophagic vacuoles, and induced robust apoptosis (51).

Autophagy inhibitors have shown clinical activity in combination with conventional therapy

Due to its superior clinical toxicity profile compared with CQ and widespread use for rheumatologic disease, HCQ has been used in most clinical trials of antiautophagic therapy for cancer (61). At maximally tolerated doses, however, the pharmacokinetics and pharmacodynamics of HCQ barely achieve concentrations required to inhibit autophagy in cancer cells. Clinical activity has not generally been observed when HCQ has been used as a single agent, but HCQ has been combined with gemcitabine for neoadjuvant therapy of pancreatic ductal adenocarcinoma (PDAC) that is at the margin of resectability, resulting in a 61% decrease in CA19–9 and a 70% complete resection rate (62). In a study of frontline therapy for stage IV pancreatic cancer, the addition of HCQ to gemcitabine and abraxane produced an increased response rate, but not an increase in overall survival (OS; ref. 63).

As a result of ongoing optimization studies to increase the efficiency and potency of CQ-derivatives, dimerization reactions have improved the cytotoxicity of aminoquinoline- and acridine-based compounds and dimeric quinacrines such as Lys05 localize more effectively to lysosomes (64). A dimeric chloroquine derivative, DC661, with an IC50 100-fold lower than that of HCQ across multiple cancer cell lines including colon and pancreas cancer cell lines has proven superior to Lys05 or HCQ in penetrating an acidic medium, blocking autophagy, and inducing cytotoxicity (65). In addition, DC661 deacidifies lysosomes and inhibits autophagic flux more effectively than Lys05, CQ, or HCQ (65). DC661 and other CQ-based drugs can target and inactivate palmitoyl-protein thioesterase 1 (PPT1), which mediates depalmitoylation to stabilize the v-ATPase subunits which not only maintain the acidity of lysosome for catabolism but contribute to the localization and subsequent activation of mTOR. Autophagy inhibitors are being developed against several other targets including ULK1, VPS34, ATG4B, and PIKFYVE (66). As more potent and selective autophagy inhibitors are being developed, an improvement in the clinical benefits that we have seen with antiautophagic therapy continues to grow proportionately.

Inhibition of RAS can enhance sensitivity to autophagy inhibitors

Autophagy is constitutively activated in several tumor types including RAS-driven cancers such as PDAC where it is essential for tumor growth and progression to scavenge intracellular nutrients (67). Paradoxically, recent evidence from different groups relates inhibition of oncogenic RAS signaling with the induction of autophagy in PDAC. Genetic suppression of KRAS, the driver oncogene in most PDAC, or pharmacologic inhibition of its effector, ERK MAPK, enhances the reliance of PDAC on autophagy (68). Dramatic antitumor activity of a combination of HCQ and MEK inhibitors has been reported in an independent study in PDAC and mouse models of other cancers driven by KRAS or BRAF mutations (69). Our preliminary data with PDAC has demonstrated that DIRAS3 induces autophagy and enhances sensitivity to the functional autophagy inhibitors CQ and HCQ (70). In addition, a synthetic peptide from the α5 region of the DIRAS3 protein produces these same effects in KRAS-mutant PDAC cells in cell culture (70). Future directions aim to delineate the molecular mechanisms by which DIRAS3 prevents KRAS-mediated signaling and activates autophagy in PDAC cells and determine the therapeutic effect of combining DIRAS3 peptides with autophagy inhibitors in preclinical xenograft models of KRAS-mutant PDAC. Thus, the initial description of PDAC addiction to autophagy is now promoting clinical progress that may lead to an effective new treatment option for patients with PDAC.

Neutralization of survival factors can eliminate dormant DIRAS3-expressing autophagic ovarian cancer cells

While upregulation of DIRAS3 induces dormancy in xenografts where cancer cells can undergo autophagy for months, reexpression of DIRAS3 induces autophagic death of ovarian cancer cells in culture within 96 hours. When factors present within the xenograft microenvironment are added to cell cultures, autophagic cancer cells can be rescued by treatment with growth factors (IGF-1 and M-CSF), angiogenic factors (VEGF and IL8), and matrix proteins (50). Survival of dormant, autophagic xenografts expressing DIRAS3 were significantly reduced and the majority of mice were cured when survival factors were neutralized with mAbs against VEGF, IL8, and insulin-like growth factor receptor (IGFR; ref. 55). Maintenance therapy with the anti-VEGF mAb bevacizumab in patients with ovarian cancer with dormant intraperitoneal metastases has increased progression-free survival (PFS) in multiple studies (71, 72). While this has been attributed to the antivascular effect of the agent, it could also relate to the removal of a critical survival factor from dormant, drug-resistant autophagic cancer cells on the peritoneum. Addition of anti-IL8 and anti-IGFR antibodies might further improve PFS or OS.

Anaplastic lymphoma kinase inhibitors eliminate dormant DIRAS3-expressing autophagic cancer cells

Despite improvements in ovarian cancer therapy, drug-resistant cancer cells survive initial therapy, often remaining dormant on the peritoneal surface for years before growing progressively to metastasize, which is a major factor contributing to poor patient outcomes. As discussed earlier in this review, the survival of dormant cancer cells in a hypovascular, nutrient-poor environment mainly depends on DIRAS3-induced autophagy and tumor dormancy in xenograft models (37, 50). A recent study identified potential targets by using unbiased siRNA screens and found that knockdown of anaplastic lymphoma kinase (ALK) reduced survival of autophagic ovarian cancer cells (73). When autophagy is induced by either upregulating DIRAS3 or serum starvation, the cell's sensitivity to ALK inhibition is enhanced significantly. The clinical relevance of this study is supported by human ovarian cancer xenograft models, where treatment of dormant, autophagic xenografts with the ALK inhibitor crizotinib produced long-term survival in a fraction of mice. Crizotinib treatment of dormant, autophagic cancer cells further enhanced autophagy and induced apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. EGFR and ABL2 have also been identified as additional potential targets which are reserved for future investigations.

Imprinted tumor suppressor genes are particularly important in cancer pathogenesis because only a single allele is expressed and loss of expression from this single allele can eliminate suppressor function, providing a “second hit.” Loss of maternally imprinted tumor suppressor DIRAS3 expression occurs through multiple genetic, epigenetic, translational, and posttranslational mechanisms, consistent with its importance as a tumor suppressor gene for a wide range of human cancers. Downregulation of DIRAS3 expression is associated with cancers from a variety of sites (Table 1). Regulation of DIRAS3 expression may, however, be far more complex. Downregulation of DIRAS3 can enhance signaling through PI3K/AKT, JAK/STAT, and RAS/MAPK. Conversely, reexpression of DIRAS3 can block malignant transformation by mutant RAS, by binding directly to RAS and disrupting RAS dimerization and clustering that is required for RAS/MAPK signaling. As DIRAS3 is expressed by humans, but not by mice or rats, animal modeling has been difficult. Therefore, there is a clear need for new systems including the patient-derived models. Based on recent advances in organoid research as well as the need to find more accurate models for target screening in cancer research, patient-derived organoids may emerge as an effective in vitro model system to study DIRAS3-mediated signaling on cancer. Positive correlations between in vitro organoid response to drugs and their matching in vivo responses have been shown in both mice and humans, patient derived systems may be helpful to address the shortage of models for studying DIRAS3. There is obvious room for improvement in terms of applicability of DIRAS3-mediated therapy. Generation of therapeutically applicable DIRAS3 peptides could provide a novel and effective method to inhibit mutant RAS function in the majority of cases for which small molecule inhibitors are not available. Strategies can also be translated from xenograft models to neutralize survival factors required by autophagic cancer cells or to repurpose drugs, such as ALK inhibitors, that are selectively toxic for DIRAS3 expressing autophagic cancer cells.

R.C. Bast reports grants from NCI during the conduct of the study. No disclosures were reported by the other authors.

This work was supported by NCI R01 CA135354 [R.C. Bast, principal investigator (PI)], the MD Anderson Ovarian SPOREs P50 CA83639 R.C. Bast and A. Sood, PIs) and P50CA217685 (R.C. Bast and Anil Sood Sood, PIs), NCI, Department of Health and Human Services; the Shared Resources of the MD Anderson CCSG grant NCI P30 CA 16672, the Cancer Prevention Research Institute of Texas RP140429 (R.C. Bast, PI), MD Anderson Cancer Center CPRIT Research Training Program Grant RP170067; and generous donations from the Ann and Henry Zarrow Foundation, the Mossy Foundation, the Roberson Endowment, the Emerson Collective, Stuart and Gaye Lynn Zarrow, Barry Elson, Arthur and Sandra Williams. Figures were created with BioRender.com.

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