MicroRNA

Abstract

Autophagy is a catabolic process that allows cellular macromolecules to be broken down and recycled into metabolic precursors. It is a highly conserved, critical process, allowing cells to gain survival advantages under various stress situations due to growth and environmental changes. In the past few years, mounting evidence indicates that the post-transcriptional and translational controls mediated by non-coding miRNAs contribute significantly to autophagy in cancer. Such acute modulation of protein synthesis mediated by miRNAs provides cells with advantages in response to starvation, genotoxic stress and hypoxia. In this review, we highlight some of the important discoveries and molecular insights of miRNAs in regulating autophagy based on various cancer models.

Introduction

In the past decade, tremendous progress has been made in understanding the molecular and cellular process of autophagy. Macroautophagy (referred to as autophagy) is an essential and highly conserved critical catabolic process that delivers cytoplasmic components to lysosomes for degradation. The process involves enwrapping fractions of the cytoplasm until a double-membrane autophagic vacuole (autophagosome) is formed. The pathways and genes, such as multiple autophagy-related genes (ATGs), involved in autophagy that have been identified in detail in recent years include ATG1, ATG4, LC3/ATG8 and beclin-1.^1,2

It is well-established that post-transcriptional and translational controls play important roles during stress situations. Such controls provide cells with acute responses to growth condition changes. Only in the past few years have we begun to appreciate the contribution and involvement of non-coding miRNAs in this process. miRNAs are non-coding RNA molecules, 18–25 nucleotides in length, which regulate the expression of their target genes by translational arrest or mRNA cleavage, most likely, through interaction mainly at the 3′-UTRs of the target mRNAs.^3-5 Base pairing between at least six consecutive nucleotides within the 5′-seed of the miRNA with the target site on the mRNA is reported to be a minimum requirement for the miRNA-mRNA interaction.^3,4 MiRNAs have been found to regulate many cellular processes, including apoptosis,^6-9 differentiation^4,10,11 and cell proliferation.^6,11-13 Deregulation of miRNAs has been associated with cancer development and progression, and miRNAs have emerged as a new research frontier for understanding cancer development at the post-transcriptional and translational level.¹⁴ Most of the past efforts of studying autophagy focused on key proteins with critical roles (first dimension) in the direct autophagy processing and the signaling pathways involved in transcriptional activation (second dimension). The contributions of post-transcriptional and translational controls (third dimension) of autophagy mediated by miRNAs emerged just recently.

MiRNAs Involved in Regulating the Expression of Key Autophagy-Related Proteins

Zhu et al. first reported the involvement of miRNA in autophagy and cancer by providing experimental evidence that miR-30a targets beclin-1, a critical scaffold protein for autophagosome formation.¹⁵ They have demonstrated that miR-30a downregulates beclin-1 expression, which mimics blunted activation of autophagy induced by rapamycin. Most recent reports further support the functional significance of miR-30a-mediated autophagy by enhancing Imatinib activity against human chronic myeloid leukemia cells.^16,17 miR-30a also sensitizes tumor cells to cisplatin by suppressing beclin-1-mediated autophagy.¹⁸ These results support a new treatment development strategy of overcoming chemoresistance by modulating miRNA-mediated autophagy.

Recently, more miRNAs have been reported to be mediators of the autophagic process. Jegga et al. proposed that miR-130, miR-98, miR-124, miR-204 and miR-142 have potential regulatory functions in the autophagic process based on computational analysis.¹⁹ Later, miR-130a was shown to inhibit autophagic flux in chronic lymphocytic leukemia (CLL) cells by decreasing the expression level of its targets, ATG2B and DICER1, which are essential for autophagosome formation.²⁰ miR-101 has been recently demonstrated as a potent inhibitor of autophagy. miR-101 suppresses autophagy induced by etoposide or rapamycin in breast cancer cells. A number of key targets, such as STMN1, RAB5A and ATG4D have been identified as direct targets of miR-101.²¹ miR-375 has been reported to inhibit autophagy through its target, ATG7, in hepatocellular carcinoma (HCC) cells.²² miR-376b expression targets ATG4C and beclin-1, which, in turn, downregulate autophagy induced by nutrition starvation and rapamycin in breast cancer cells.²³ On the other hand, ectopic expression of miR-7 in human lung cancer and esophageal cancer cell lines enhances autophagy by suppressing epidermal growth factor receptor (EGFR) expression.²⁴

We have recently identified that miR-502 directly suppresses autophagy by decreasing the expression of RAB1B in colon cancer cell lines.²⁵ RAB1B is a small GTPase from Ras super family that has been demonstrated to modulate autophagic activity in HeLa cells through the regulation of autophagosome formation.²⁶ Rab1B has been shown to regulate vesicle trafficking at multiple stages and directly impact autophagy^27,28 and was found to be overexpressed in liver cancer.²⁹ Ectopic expression of miR-502 in HCT-116 cells interrupted autophagic flux under acute and prolonged nutrient starvation.²⁵

Besides the contribution to cell proliferation or cell death, drug-induced autophagy also has been shown to play an important role in cancer chemoresistance, especially for cisplatin.^30-32 Cisplatin treatment activates autophagy in multiple cancer cells and decreases the level of several miRNAs at the same time, which target multiple key regulators in autophagy, such as miR-199a-5p against ATG7 in HCC,³³ miR-181a and miR-374a against ATG5, miR-630 against ATG12 and miR-519a against beclin-1, ATG10 and ATG16 in squamous cell carcinoma (SCC) cells.³⁴

The miRNA-mediated genes and pathways involved in autophagy are illustrated in Figure 1.

Figure 1. The miRNA-mediated genes and pathways involved in autophagy.

MiRNAs mediated autophagy in genotoxic stress and hypoxia

Evidence suggests that autophagy plays a prominent role in allowing cells to survive under stressful conditions such as hypoxia. While the exact role autophagy plays in cancer development remains uncertain, it is clear that the induction of autophagy in tumor cells helps them survive the hypoxic conditions associated with the inadequate blood supply to the tumor. It has been shown that miRNAs can mediate this autophagic response to hypoxia. In particular, miR-375, normally downregulated in HCC when exogenously expressed, was shown to inhibit autophagy in response to hypoxia by targeting ATG7, reducing the conversion of LC3I to LC3II. In vivo mouse xenograft studies demonstrated that miR-375 expression reduced tumor growth compared with controls.³⁵ Similar results were found looking at hypoxia in cardiomyocytes. Jian et al. found that miR-204 is downregulated by hypoxia-reoxygenation treatment, but when overexpressed, reduced the conversion of LC3I to LC3II.³⁶ These findings lend support to the idea that the regulation of autophagy by miRNAs may be a viable target for treatment of cancer, making tumor cells less resistant to the inherited hypoxic conditions of the tumor or more susceptible to the effects of other drugs. The story is complex, however, as other miRNAs have been shown to play a role in the activation of autophagy. In Cav(−/−) breast tumor stromal cells, miR-31 and miR-34 were both found to be upregulated. These miRNAs are associated with oxidative stress response and activation of autophagy through HIF1α, promoting cancer cell survival.^37,38 miR-34 has also been implicated in the response to DNA damage through both p53-dependent and -independent mechanisms.³⁹ Much like the role of autophagy in cancer development, the role that miRNAs play in cancer development and autophagy regulation is complex. The complexity is most likely influenced by the unique tumor microenvironment. Much work needs to be done to better elucidate the dynamic role of miRNAs in the regulation of autophagy in response to cellular stress, as well as the effects that this response has on the cell.

MiRNA, Cell Cycle Control and Autophagy

Because one particular miRNA can regulate a number of target mRNA transcripts to control their translation, this provides cells with greater flexibility to utilize miRNA in response to growth condition changes and to different environments. This has been recently demonstrated for miR-10b. In breast cancer, miR-10b promotes breast tumor invasion and metastasis by suppressing homeobox D10 (HOXD10).⁴⁰ In contrast, miR-10b has been demonstrated to promote cell proliferation and prevents death of glioblastoma cells by targeting cell cycle inhibitors and pro-apoptotic genes.⁴¹ miR-34c has been shown to regulate the cell cycle in response to DNA damage through its regulation of c-Myc.³⁹ Following DNA damage, miR-34c, activated either by p53 or p32 MAPK/MK2, represses c-Myc, preventing further DNA synthesis, arresting the cells in S-phase.³⁹ We have also demonstrated the flexibility of miRNA to regulate both the cell cycle and autophagy. We showed that miR-502, through its effect on different targets, can both regulate autophagy and cell cycle arrest at both the G₁ and G₂ phase. Overexpression of miR-502 in HCT-116 cells both inhibited autophagy and increased the number of cells in G₁ and G₂ phases in comparison to S phase.²⁵

MiRNA: The Third Dimension of p53-Mediated Autophagy

Cisplatin has been shown to induce autophagy in cancer cells^33,34 and activate p53 family members,^42,43 which, in turn, function as transcriptional regulators to affect downstream gene expression, including miRNAs.^44-46 In SCC cells, cisplatin exposure phosphorylates ΔNp63α, which directly regulates the transcription of many miRNAs.⁴⁴ Among the direct targets, miR-181a, miR-519a, miR-374a and miR-630 has been shown to regulate autophagy-related proteins,³⁴ which suggests that miRNAs can modulate autophagy pathway at another translational level.

A number of previous studies have reported the direct involvement of p53 in autophagy. The cytoplasmic pool of p53 suppresses autophagy under nutrient deprivation. Moreover, p53 also functions as a key regulator of autophagy.^47-50 Under prolonged nutrition starvation, p53 sustained active autophagic flux, which is beneficial for colon cancer cell survival.⁵⁰

Since translational control provides cells with acute responses to growth stress, we reasoned that some of the p53-mediated miRNAs may play important roles in autophagy. We have provided evidence that certain miRNAs (miR-502) are also involved in the p53-mediated autophagy pathway.²⁵ Ectopic expression of miR-502 in colon cancer cells decreases p53 and upregulation of p53 triggers downregulation of miR-502, suggesting a negative feedback loop between p53 and miR-502. Moreover, overexpression of miR-502 in wild-type p53 cells leads to stalled autophagic flux, and this phenomenon is more prominent in p53-null cells, indicating miR-502 has a broader impact on autophagy that is beyond p53. The impact of miR-502 on autophagy pathway is schematically demonstrated in Figure 1.

Therapeutic Potential of miRNAs and Autophagic Pathway

There is mounting evidence to link autophagy and cancer,^51,52 and the role of autophagy in cancer progression is still unclear. Several reports demonstrated that activation of autophagy may suppress tumor development and lead to cell death.^53,54 However, it has been demonstrated extensively that autophagy mediates tumor survival by supplying nutrients to stressed cancer cells.⁵⁵ Therefore, an anti-autophagy approach may also offer a therapeutic strategy for treating cancer.^54,56,57 One of the examples is rapamycin and its analogs (e.g., RAD001), which inhibit a kinase-named mammalian target of rapamycin (mTOR). Moreover, it has been demonstrated that inhibition of autophagy by 3-methyladenine or siRNA against Atg7 enhances the apoptosis induced by 5-FU treatment in colorectal cancer cell lines in vitro and in vivo.⁵⁸ In Ras-driven tumors, it has shown that Ras mediates cell transformation and activates autophagy, which is essential for tumor cell survival.⁵⁵ Inhibition of autophagy in Ras-expressing cells leads to decreased cell proliferation, transformation and failure of tumor formation in mice.^59-61 Contrary to this, other studies have shown an anticancer role by enhancing autophagy.^47,52,62,63Table 1 provides a list of miRNAs reported with therapeutic potential for treating cancer. It seems clear that the unique oncogenes/pathways involved in autophagy, the specific tumor type and tumor environment and the disease context are some of the key factors to be considered for developing autophagy-mediated anticancer strategies.

Table 1. miRNAs reported with therapeutic potential for treating cancer.

miRNA	Targets	References
miR-101	STMN1, RAB5A, ATG4D	Frankel LB et al., 2011
miR-221/222	p27	Miller TE et al., 2008
miR-30a	Beclin-1	Zou Z et al., 2012
miR-375	ATG7	Chang Y et al., 2012
miR-502	p53, Rab1B	Zhai H et al., 2012

In the case of miRNA, a number of reports have shown that miRNA mimics or anti-miRs can be used as potential therapeutics for anticancer drug development. Breast cancer cells treated with miR-101 showed increased cell death mediated by 4-hydroxytamoxifen (4-OHT) due to the inhibition of autophagy,²¹ suggesting that microRNAs may modulate chemosensitivity of cancer cells. miR-30a also enhances the tumor cell apoptosis induced by cisplatin through suppressing belcin-1-mediated autophagy.¹⁸ miR-221/222 expression on the other hand has been shown to increase tamoxifen resistance in breast cancer by decreasing levels of p27.⁶⁴ Similarly, miR-21 expression level is found to be higher in radio-resistant glioblastoma cells than in radio-sensitive ones.⁶⁵ Inhibition of miR-21 can increase the radio-sensitivity of glioblastoma cells and enhance the apoptosis after radiation through upregulate autophagic activity.⁶⁵

We have demonstrated that miR-502 can significantly decrease colon cancer cell growth in vitro and induce cell cycle arrest. Inhibition of both p53 and RAB1B, the mediators for autophagy, can reproduce this phenotype, suggesting that autophagy plays an important role in its tumor-suppressive function. Profiling of human colon cancer samples reveals that miR-502 is downregulated in tumor tissue as compared with normal tissue. Ectopic expression of miR-502 in human colon cancer xenografts can significantly reduce the tumor sizes, indicating miR-502 as a potential adjuvant treatment for colon cancer patients.²⁵

Conclusion and Future Perspectives

It is clear that miRNAs contribute to autophagy under various stress situations, especially in cancer. As cancer cells grow under stress conditions due to hypoxia, miRNAs will impact cancer cell survival by modulating autophagy. Therefore, it is conceivable that modulating miRNA will open a new direction to change cancer cells in response to stress by altering the autophagy process and, in turn, will provide new therapeutic strategies to overcome chemoresistance.

Glossary

Abbreviations:

ATG

autophagy-related gene

CLL

chronic lymphocytic leukemia

EGFR

epidermal growth factor receptor

HCC

heptocellular carcinoma

4-OHT

4-hydroxytamoxifen