Cancer genesis and lncRNA: Developments from 2012

Vy Nguyen

Keywords
lncRNA, cancer therapy, misregulation, tumorigenesis, tumor suppressing proteins

Abstract
The most common cancer therapies, chemotherapy, radiation, and surgery, are generalized and often ineffective. As a result, scientists have been searching for more targeted drugs to treat tumors. New studies have focused on long non-coding RNA (lncRNA), non-coding RNA molecules greater than 200 nucleotides in length. lncRNA has long been associated with essential cell functions, such as control of gene splicing, translation, and transcription. lncRNA has also been linked to cancer, with evidence showing that cancer cells often exhibit abnormally high or low levels of lncRNA. Recent studies from 2012 have focused on defining the specific molecular pathways connecting lncRNA and tumorigenesis. By discovering the tumor-suppressing proteins linked to misregulated lncRNA, researchers have been able to propose possible cancer treatments involving the artificial regulation of lncRNA. This literature review outlines recent findings in lncRNA and cancer and identifies potential applications for the future.

Introduction
Chemotherapy, the most widely used treatment against cancer, is designed to kill rapidly dividing cells in the body. While cancer cells fit under this category, beneficial cells such as skin, hair, stomach, and blood cells do as well. As a result, cancer patients undergoing chemotherapy experience side effects such as loss of hair, cognitive impairment, and nausea.1 In addition, chemotherapy is often ineffective and only able to put cancer temporarily at bay. Because chemotherapy is such a generalized treatment, scientists have been searching for a more specialized drug to treat cancer. Cancer, they have discovered, is caused by the misregulation of growth factors in a cell.2 For example, cancer cells do not properly respond to signals limiting cell growth and promoting cell death and differentiation. However, the cause of this misregulation has been linked to a whole diversity of factors, including chemical exposure, abnormal proteins, and viruses.3 In order to dive closer to the root of the cause, recent studies have focused on non-coding RNA (ncRNA.) Non-coding RNA, though they do not translate into proteins, are key in regulating gene expression in the cell.4 Abnormalities in ncRNA activity could mean irregular growth in cells that lead to ongogenesis. Scientists have found evidence linking a certain type of noncoding RNA, known as long non-coding RNA, to various types of cancer.

lncRNA: A new kind of molecule

Non-coding RNA molecules perform several functions in the cell, including the regulation of gene expression, post-replication telomeric repair, and control of gene splicing.4 One of these recently discovered RNA

molecules is long non-coding RNA (lncRNA). lncRNA, broadly defined as non-coding RNA molecules that are 200 nucleotides or greater in length,5 can be present in the thousands within a single cell. While lncRNA was at first believed to be transcriptional junk,6 new research has shed light on the important role lncRNA plays in cells.

The role of lncRNA in cells

lncRNA is an important regulator in many cell processes. For one, lncRNA controls gene expression during all stages of transcription. At the pre-transcription level, they can modify transcription factor activity or serve as co-regulators. Post-transcriptionally, lncRNA has a role in gene splicing, translation, and mRNA silencing.5 lncRNA is even involved in epigenetic regulation.7 For instance, lncRNA seems to control genetic imprinting, in which the alleles of only one parent is expressed in an individual despite the offspring possessing alleles from both parents. Similarly, lncRNA is a factor in inactivating the second X-chromosome in females. lncRNA has also been linked to several illnesses, including Alzheimers disease and Huntingdons disease.8 The specific lncRNA transcripts involved in essential molecular pathways are highly evolutionary conserved across several species, further suggesting their importance.7

lncRNA in cancer
lncRNA appears to play a part in carcinogenesis, making it the subject of several recent studies. Cancer cells frequently exhibit abnormally high or low levels of lncRNA. Recently, researchers have uncovered several pathways between which lncRNA and cancer are linked. This section outlines some of these ways that lncRNA contributes to tumor formation and growth.

Upregulation
The term upregulation refers to the increased transcription of a specific RNA molecule in a cell so that it is present in larger quantities. In some cases, upregulation of certain lncRNA transcripts leads to the greater proliferation of cancer cells. For instance, the lncRNA highly-upregulated in liver cancer (HULC) is, as the name suggests, present in abnormally high amounts in heptatocellular carcinoma (HCC).9 Gastric cancer cells have exhibited high levels of H19, another lncRNA transcript.10 In these cases, the lncRNA appears to interfere with the creation of tumor suppressor proteins. For example, HBx, a hepatitis B viral protein, causes the upregulation of HULC by interfering with the CREB transcription factor. The altered CREB factor causes increased transcription of HULC. HULC, in turn, downregulates p18, a tumor suppressor gene. Because the gene was silenced, less p18 proteins were being produced, and thus the HCC cells were was allowed to grow and migrate. H19, on the other hand, is linked to gene p53. p53 prevents tumor formation by promoting cell death and preventing cell proliferation. H19 partially inactivates the p53 gene, which in turn causes lower amounts of p53 target protein Bax to be produced in the cell. Without Bax, the cell can replicate without restraint, causing the gastric tumor. In fact, one of the first lncRNA transcripts discovered, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), has been linked to multiple types of cancer (see table 1, row 3).11 MALAT1 is actually highly expressed in healthy cells. It is an important regulating factor in cell migration, formation, and apoptosis.3 However, cancer tumors have shown excessive levels of MALAT1 expression. It is not the mere presence of these lncRNA molecules that causes tumors to grow. Rather, it is the misregulation of the transcripts that is the cause.

lncRNA transcript HULC H19 MALAT1 MEG3 LOC554202

Cancer type HCC Gastric HCC, adenocarcinoma, breast Glioma TNCB

Down or upregulated Up Up Up Down Down

Associated protein p18 p53 Unknown p53 p53

P-value P < 0.0001 P < 0.05 P = 0.012 P < 0.05 P < 0.05

TABLE 1: A comparison of cancer-linked lncRNA transcripts This table compares differences and similarities between several lncRNA transcripts by cancer type, misregulation, associated tumor suppressing protein, and p-value. Note that MALAT1 is capable of affecting several types of cancer, and that p53 is linked to multiple regulatory lncRNAs.

Downregulation
The upregulation of lncRNA is not always associated with cancer tumorigenesis. Downregulation of lncRNA can also cause tumor growth. In brain tumor cells, the expression of lncRNA maternally expressed gene 3 (MEG3) is actually decreased.12 MEG3 regulates cell proliferation by association with tumor suppressor gene p53. When MEG3 is not expressed, p53 is also not expressed, allowing the glioma cells to grow uncontrollably. The lncRNA molecule actually has a tumor suppressing effect. The connection between lncRNA and the tumor suppressing protein can be indirect. The novel lncRNA LOC554202 is the host gene to micro RNA miR-31, a tumor suppressing gene involved in triple-negative breast cancer (TNCB).13 TNCB cells show abnormally low levels of both LOC554202 and miR-31, suggesting their connection. miR-31 then suppresses several metastasis-promoting genes, including RhoA and WAVE3, which are linked to p53. LOC554202, in this case, is only a part of a long chain of gene interactions, which could be detrimental when developing TNCB treatments based on LOC554202. For instance, LOC554202 and miR-31 appear to be regulated epigenetically via CpG island hypermethylation. When researchers altered the DNA methylation in an attempt to upregulate LOC554202 and miR-31, the miR-31 levels did not reach those of the lncRNA host transcript. This could possibly be attributed to other factors regulating mi-R31, making LOC554202 an ineffective target for therapy.

Cancer therapy applications

Researchers have begun testing the possibility of using lncRNA-based strategies in cancer medicine. First, lncRNA could be used to distinguish between various types of cancer. Second, the manipulation of lncRNA expression could become an important tool in reducing the growth of tumors.

lncRNA as a diagnostic tool

As almost every type of cancer exhibits some sort of lncRNA misregulation, lncRNA could become a valuable biomarker in the future. lncRNA expression can be detected in blood and urine samples by ordinary PCR methods,5 making it a potentially low-cost and convenient tool in cancer detection. New studies are already taking advantage of this fact. For example, lncRNA markers can differentiate between different types of brain tumor cells.14 There are two main types of brain tumors, astrocytomas and oligodendrogliomas. It is difficult to distinguish between these different types by inspecting the anatomy of the cells alone. However, astrocytomas and oligodendrogliomas exhibit unique patterns of lncRNA expression, offering a possible method to distinguish between them. Because the two types respond differently to cancer

therapy, being able to identify the type of glioma could help oncologists tailor a more effective, personalized treatment for their patient. However, at this time, the lncRNA expression patterns for other types of tumors are poorly characterized. Future work in this area could bring lncRNA analysis closer to practical clinical application.

Manipulation of lncRNA expression

Interfering with lncRNA expression may be able to stop a tumor at the cause, opening the possibility for lncRNA-based cancer drugs. For instance, as described above, the upregulation of HULC leads to tumor growth. However, HULC can be artificially downregulated using CREB siRNA.9 The CREB siRNA blocks the HULC promoter, preventing Hbx proteins from activating transcription. Knockdown of HULC causes upregulation of p18, which in turn stops the proliferation of HBx-enhanced cancer cells. Similarly, MALAT1 can be downregulated via siM1 and siM2.11 After 48 hours of MALAT1 knockdown, HCC cells show reduced proliferation. The cells are also more sensitive to apoptosis signals such as doxorubicin and cisplatin. Treatment of cells with a specific siRNA could reduce tumors that show unusually high levels of a certain lncRNA. Because MEG3 is already downregulated in carcinomas, treatment with siRNA is not effective as it is for HULC and MALAT1. However, MEG3 can be upregulated in glioma cells through treatment with the p53 antibody or by transfection with the p53 plasmid and MEG3.12 Although the precise mechanism is still unclear, upregulation of MEG3 suppresses tumor growth. While methods may vary, artificially restoring an lncRNA transcript to its normal level can have a tumor suppressing effect.

Conclusion
lncRNA has long been associated with cancer genesis. However, the molecular pathways between cancer cells and lncRNA are only beginning to be understood. Research suggests that misregulation of lncRNA whether through down or upregulationcould be the root of the cause of cancer. Each new discovery brings another potential route for targeted cancer therapy by manipulation of lncRNA expression. In addition, unique lncRNA expression patterns between cancer cells could help differentiate between various types of tumors. lncRNA-based treatment could be a future path for clinical oncology.

Acknowledgements
I would like to thank my peer reviewers, Caitlin Morelli and Dylan Kaufman, for their invaluable comments and critiques. I would also like to thank Dr. Cecelia Musselman for her input on the grammar and missing points of this review. Finally, I would like to thank Shailja Modi for proofreading this review for spelling and grammatical errors. Without these people, this paper would not have been possible.

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