Advanced Review
The role of microRNA in resistance to breast cancer therapy Neil M. Robertson1 and Mehmet V. Yigit2∗ MicroRNAs (miRNAs) are small noncoding RNA molecules with big implications in cancer. The abnormal expression of specific miRNAs has been linked to development of many cancer types. Dysregulated miRNAs play a significant role in proliferation, invasion, differentiation, apoptosis, and resistance of various cancer cells, and considered as oncogenes or tumor-suppressor genes. Findings have shown abnormal expression of specific miRNAs in breast tumors is a strong indication about the resistance to conventional cancer therapy methods. Acquired cancer resistance is a complex, multifactorial occurrence that requires various mechanisms and processes, however, recent studies have suggested that resistance may be linked to treatment-induced dysregulation of miRNAs. This dysregulation of miRNAs can affect the protein expression in cells, the ability for anti-cancer drugs to reach their targets within cells, and the apoptotic pathways. Controlling the expression of these miRNAs alters the resistant phenotype of breast cancer to a nonresistant one. This review focuses on the role of dysregulated miRNAs in breast cancer that are linked to resistance against chemo-, radiation, hormone, and targeted therapies. Finally, the role of miRNAs in breast cancer metastasis is briefly discussed. © 2014 John Wiley & Sons, Ltd. How to cite this article:
WIREs RNA 2014. doi: 10.1002/wrna.1248
INTRODUCTION
M
icroRNAs (miRNAs) are endogenously expressed small noncoding RNA molecules (∼22 nt), which are involved in posttranscriptional regulations of numerous different target messenger RNAs. They bind to the 3′ untranslated region (UTR) of the mRNAs and downregulate target proteins by translational repression or by degradation of mRNAs. miRNAs play a critical role in several stages of various cancer types.1 They are involved in multiple cellular pathways, including cell proliferation, invasion, differentiation, and apoptosis, and act as oncogenes or tumor suppressor genes. The deregulated expression of certain miRNAs in cancer cells has been a strong ∗ Correspondence 1 Department
to: [email protected]
of Chemistry, University at Albany, SUNY, Albany,
NY, USA 2 Department of Chemistry and RNA Institute, University at Albany,
SUNY, Albany, NY, USA Conflict of interest: The authors have declared no conflicts of interest for this article.
indication for different cancer types and the disease stage.2–14 Controlling the expression of these abnormally expressed miRNAs has resulted in successful therapeutic outcomes in cultured cancer cells and animal models.15–17 miRNAs are considered as important biomarkers and therapeutic targets for cancer.18 It has been shown that many dysregulated miRNAs play critical roles in breast cancer, which is the most common cancer among American women and one of the leading causes of cancer related deaths in women worldwide.19–25 According to the American Cancer Society, death rates from breast cancer have been declining for the past two decades due, in part, to improved treatments along with earlier detection and implementation of screening programs. The desire for early detection and screening programs allows for the identification of miRNA signatures associated with different breast tumors, not only for therapeutic options, but also for better diagnosis. While the improved treatments have led to our current methods of chemotherapy, radiation therapy, hormone therapy, and targeted therapy. However, resistance to these
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therapy methods could develop after a period of time, which decreases the efficacy of the treatment dramatically. It has been demonstrated that abnormal expression of specific miRNAs has strong implications in the resistance against these therapy methods.26–31 Chemotherapy is a treatment which relies on drugs to inhibit the growth and progression of the tumor and to induce cancer cell death. Chemotherapeutic anti-cancer agents (1) can be taken orally or by injection to enter the bloodstream and circulate throughout the body, known as systemic chemotherapy, or (2) can be applied directly into an area for effect, known as regional chemotherapy. The administration depends on the cancer type and stage, however, both are effective means of chemotherapy. Chemotherapy is often used for cancer that has occurred at multiple organs or spread to the other tissues. Chemotherapy is effective, but also carries the problem of side effects due to the death of healthy cells along with cancer cells because of the nonselectivity of the drugs. Another serious problem is the acquisition of drug-resistance in the cancer cells over time. Findings have shown miRNAs to play a key role in drug-resistance, intricately controlling the appearance of the drug-resistant phenotype by their aberrantly expressed levels.32–34 Despite the problems, chemotherapy remains an effective approach in the cancer treatment process. Radiation therapy, radiotherapy, is another mainstay treatment method that uses different types of radiation to inhibit the progression of cancer. With two different types of radiation therapy, external radiation and internal radiation, the choice of therapy is again dependent on the cancer type and stage. Though an effective means of destroying cancer cells, radiation therapy is best used against a single tumor or mass. Much like chemo-resistance, radio-resistance appears in cancer cells after prolonged exposure to the treatment. It has been described that miRNA dysregulation in these cells is, at least in part, responsible for the radio-resistance in the breast tumors.35–40 Hormone therapy is the treatment that blocks or reduces the hormone levels to inhibit the progression of cancer cells. Since some hormones are known to cause certain cancers to grow, blocking these hormones greatly slows the progression of the disease and increases the chance of disappearance of the tumor. While a good treatment, hormone therapy is limited to very few cancers, breast cancer included as one, and is only able to slow the progression. It must be combined with another therapy to induce cancer cell death. Along with the selectivity of the treatment, the acquired resistance to the hormone over time due
to miRNA dysregulation remains problematic in its continued use.41,42 Lastly, targeted therapy is performed using drugs or sometimes small molecules to identify and target tumors without damaging the healthy tissues. Within targeted therapy, multiple techniques including monoclonal antibody therapies and tyrosine kinase inhibitors are found. Monoclonal antibody therapy is administered by infusion and utilizes antibodies to target molecules necessary for the cancer cells to survive. By attaching to the specific molecules, they block the growth rate of the cancer cells, prevent them from spreading, or even induce cancer cell death. Tyrosine kinase inhibitors are utilized similar to block the signals necessary for the malignant cells to grow. While powerful techniques, the therapies have only been approved for use in specific cancer types and are usually applied along with another treatment, such as chemotherapy. Also, a recurring theme is seen with prolonged treatment, the patients develop resistance to it and often another targeted therapy to overcome this resistance is not available. This acquired resistance to targeted therapies is caused, in some cases, by the irregular expression of miRNAs.28 miRNAs can act as oncogenes—oncomiRs—or tumor-suppressor genes by either repressing translation or inducing specific mRNA cleavage.1 The above cancer treatments all share a few traits, one of which being the abnormal expression of miRNAs playing a role in the problems arising from continual use of the treatments. The studies discussed in this review were performed on tumor samples, cultured breast cancer cells or animal models. The list of miRNAs found to play a role, and the effects they bring, is continuously expanding as new reports are published. Their role in other aspects of cancer is also being well studied and there are numerous reports on miRNAs role in breast cancer metastasis and proliferation which is only briefly discussed in this review article.24
miRNAs’ ROLE IN RESISTANCE TO CANCER THERAPY The dysregulation of miRNAs not only plays an important role in cancer development and progression, but has also been found to intricately control the acquisition of drug-resistance and, as a result, the overall success of cancer therapies. Acquired cancer resistance is a complex, multifactorial occurrence that requires various mechanisms and processes, however, recent studies have suggested that chemo-resistance may be linked to drug-induced dysregulation of miRNAs.43 This dysregulation of miRNAs can affect the protein expression in cells,
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The role of miRNAs in resistance
the ability for anti-cancer drugs to reach their targets within cells, and the apoptotic pathways. The past and ever increasing present studies have demonstrated that miRNAs confer resistance to all currently used treatments against breast cancer.
Chemotherapy It has been known that certain proteins [multidrug resistance protein 1 (MDR1)/p-glycoprotein, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP/ABCG2)] play a role in the chemo-resistance of breast cancer cells. As more studies are published, findings are pointing toward the expression of miRNAs being a key regulator of these proteins and their function in the resistance to specific anti-cancer drugs.
Doxorubicin Resistance With doxorubicin (also known as Adriamycin) being a commonly used drug in chemotherapy, the acquired resistance, due to miRNA up- or downregulation, becomes problematic with its continued use in treatment. However, recent studies have shown that altering the levels of the miRNAs in the cells can lead to a promising re-sensitization of the cells. For example, the miRNA-200 family (miR-200b, -200c), specifically the upregulation of them, regulates the transcription of E-cadherin and leads to an overall reversion to a less aggressive cancer phenotype and an increased sensitivity to doxorubicin in resistant cancer cells.44 Similarly, another study demonstrated that the levels of miR-128 in breast tumor-initiating cells to be significantly reduced and accompanied by an over expression of the resistant proteins BMI-1 and ABCC5. Ectopic expression of miR-128 reduced these protein levels, decreased cell viability, and increased cell apoptosis and DNA damage in the presence of doxorubicin.33 Just as increasing the expression of the miR-200 family and miR-128 increased sensitivity to doxorubicin, other studies showed that increased expression of miR-29845 and miR-32632 was found to regulate the doxorubicin chemo-resistance in other cell lines. In cells resistant to doxorubicin, the miR-298 levels were reduced, which lead to an increase in p-glycoprotein and prevented doxorubicin from reaching the nucleus of the cells. The overexpression of miR-298 reversed this effect, with an increased nuclear accumulation of doxorubicin and cytotoxicity observed.45 Likewise, downregulation of miR-326 created a resistant phenotype and when the levels were elevated, the cell lines became sensitized to a doxorubicin treatment.32 Reduced expression levels of miRNAs seem to be a common trend with
doxorubicin resistance in breast cancer cell lines, however, it is not always the case. In MCF-7/Adr resistant cells, five upregulated miRNAs (miR-100, miR-29a, miR-196a, miR-222, and miR-30a) were classified as potential causes for the resistant phenotype. Results showed it was miR-222 and miR-29a that played the largest role, with mimics and inhibitors partially changing the drug-resistance of breast cancer cells.46
Taxane Resistance Other very well known, and commonly used, drugs in the treatment of breast cancer are Paclitaxel (Taxol) and Docetaxel. miRNA dysregulation has recently been found to play a key role in the level of resistance to these core treatment drugs. miR-125b upregulation in taxol-resistant cells caused an inhibition of taxol-induced cytotoxicity and apoptosis.47 This was due to miR-125b suppression of the pro-apoptotic protein Bcl-2 antagonist killer 1 (Bak1), found to be a direct target of the miRNA. Inhibition of miR-125b (or increasing the levels of Bak1) overcame the miRNA-mediated taxol resistance and recovered sensitivity to taxol. Another study showed that expression of miR-125b in vivo led to increased resistance and a more advanced carcinoma in patients, shown in Table 1. miR-125b may be an efficient biomarker to determine effectiveness of treatment and also a suitable target to affect Taxol sensitivity, (Table 1).48 Other miRNAs, miR-29a and miR-222, previously stated to play a role in doxorubicin resistance, were also found be at least partially responsible for acquired resistance to Docetaxel. Mimics and inhibitors of miR-29a and miR-222 were found to alter the drug-resistance of the breast cancer cell line.46 However, another study showed they were not the only miRNAs found to affect Docetaxel resistance.49 The resistance was associated with increased expression of miR-34a and miR-141 and decreased expression of miR-7, miR-16, miR-30a, miR-125a-5p, and miR-126, some of the many miRNAs found dysregulated are organized in Table 2.49 The response to Docetaxel in resistant cells was found to be enhanced with an inhibition of miR-34a.
Mitoxantrone Resistance With Mitoxantrone being used for treatment of metastatic breast cancer, there is no surprise that increasing sensitivity to the drug could improve the survival rate in breast cancer patients. miR-328 was found to negatively regulate the expression of the extremely important breast cancer resistance protein (BCRP/ABCG2) in human cells.50 There is an inverse relationship between miR-328 and BCRP/ABCG2
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TABLE 1 miR-125b Was Found to Be a Key Biomarker for the Success of Treatment in Breast Cancer Patients. (Reprinted with permission from Ref 48. Copyright 2012 Public Library of Science; PLOS) M (SD)
n (total 56)
miR-10b
miR-34a
miR-125b
miR-155
35 (63%)
1.5 (0.7)
3.5 (1.6)
**2.2 (0.4)
*0.9 (0.2)
2.2 (1.1)
5.1 (2.6)
**6.8 (2.1)
*3.1 (0.6)
1.7 (0.6)
3.8 (1.9)
*2.6 (0.7)
1.5 (0.4)
2.1 (1.0)
4.9 (1.2)
*6.1 (1.9)
2.3 (1.0)
1.9 (0.9)
3.9 (1.3)
3.6 (1.1)
1.7 (0.5)
Characteristics
Subgroups
Clinical stage
II III
21 (38%)
Grade
II
32 (57%)
III
24 (43%)
Distant metastasis
M0
20 (36%)
M1
36 (64%)
2.1 (0.9)
4.5 (2.1)
5.9 (1.8)
2.1 (1.2)
Node metastasis
N1-3
40 (71%)
1.5 (0.7)
3.2 (1.1)
*3.1 (0.5)
1.9 (1.0)
N≥4
16 (29%)
2.3 (1.2)
5.2 (2.6)
*6.3 (1.1)
2.1 (0.9)
Negative
13 (23%)
2.1 (1.1)
4.1 (1.9)
4.6 (1.3)
2.2 (1.1)
ER
Positive
43 (77%)
1.9 (0.8)
4.7 (1.9)
4.1 (1.1)
1.9 (0.6)
PR
Negative
19 (34%)
1.9 (0.5)
3.9 (1.5)
3.9 (0.9)
1.6 (0.5)
Positive
37 (66%)
2.2 (1.3)
4.6 (2.1)
4.8 (1.5)
2.3 (1.2)
HER2
Negative
46 (82%)
1.7 (0.8)
3.8 (0.9)
3.8 (1.4)
2.1 (0.9)
Positive
10 (18%)
2.0 (1.2)
4.5 (1.3)
4.2 (1.8)
1.7 (1.1)
ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor. Upregulation of miR-125b led to increased resistance and a more advanced carcinoma, poor characteristics in advanced stages of breast cancer. Patients’ characteristics at the time of primary diagnosis of breast cancer and correlation with serum miRNA levels. *p < 0.05, **p < 0.01.
with miR-328 upregulation resulting in an increased Mitoxantrone sensitivity in MCF-7/MX100 cells.50
Cisplatin Resistance Cisplatin was the first member of a class of platinum-containing anti-cancer drugs and an important chemotherapy drug to trigger apoptosis. In MCF-7 breast cancer cells resistant to cisplatin, a large number of dysregulated miRNAs were discovered. The most significantly dysregulated were miR-146a, miR-10a, miR-221/222, miR-345, miR-200b, and miR-200c while miR-345 and miR-7 were found to target the human multidrug resistance protein 1, MDR1.43
Multidrug Resistance Not all miRNAs seem to affect a specific drug alone or maybe the miRNAs above have not yet been fully explored as to which resistances they confer, regardless, there are many newly found miRNAs that are responsible for a multidrug resistance in human breast cancer cells. For example, a recent study showed that miR-505 inversely correlates to Akt3 and modulates drug sensitivity in the MCF-7-ADR cell line.51 Another study demonstrated that the production of miR-21 lead to a decrease in the tumor-suppressor protein PDCD4, which resulted in the upregulation of inhibitors of apoptosis proteins and MDR1. They used a specific anti-miR-21 inhibitor to silence miR-21
expression and enhance PDCD4 expression, which blocked the mediated cell behavior.34 Other miRNAs to take note of their regulation are miR-155, where the knockdown rendered cells vulnerable to apoptosis and enhanced chemo-sensitivity,52 and miR-19, where inhibition restored sensitivity to cytotoxic agents in multidrug-resistant cells.53
Radiotherapy Radiation therapy is an extremely powerful tool in the treatment of breast cancer and is often highly utilized alongside chemotherapy. The acquired resistance found in breast cancer cells after prolonged exposure has led to problems and in some cases the failure of the treatment. Therefore, the discovery of miRNA dysregulation playing a key role in the radio-resistance was an important step for future clinical work. One miRNA that was found to be abnormally expressed in irradiated breast cancer cells was miR-302.36 The authors found miR-302 to be downregulated and replacement therapy re-sensitized the cells to radiation therapy in vitro and in vivo, see Figure 1.36 In this study, mice xenografts, resistant to radiation, were transfected with miR-302 or controls and then dosed with radiation to kill cells, 5 Gy radiation, or a mock level, 1 Gy radiation. As depicted in Figure 1, the controls remained resistant to radiation and showed no loss in tumor size and miR-302 replacement
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TABLE 2 Docetaxel Resistance in Cancer Cell Lines Was Found to be Associated With Increased or Decreased Levels of miRNAs. Results represent mean fold changes based on duplicate experiments. A miRNA expression in docetaxel-resistant cells compared to docetaxel-sensitive cells, where sensistive cells have a fold change of +1. Decreased miRNA expression is indicated with a − (minus); increased miRNA expression is indicated with a + (plus). (Reprinted with permission from Ref 49. Copyright 2012 Springer)
Cell Line MCF-7
MDA-MB-231
miRNA Expressiona
Fold Change (±SD)
P Value
hsa-miR-7
−
−3.87 ± 0.08
0.018
hsa-miR-16
−
−2.64 ± 0.01
0.034
hsa-miR-30a
−
−2.03 ± 0.21
0.010
hsa-miR-30d
−
−2.58 ± 0.32
0.048
has-miR-125a-5p
−
−1.88 ± 0.11
0.042
hsa-miR-126
−
−2.96 ± 0.55
0.025
hsa-miR-129-3p
−
−6.95 ± 4.51
0.006
hsa-miR-138
−
−6.29 ± 0.61
0.045
hsa-miR-149
−
−2.24 ± 0.18
0.023
hsa-miR-151-3p
−
−2.37 ± 0.16
0.007
hsa-miR-151-5p
−
−2.79 ± 0.46
0.009
hsa-miR-190b
−
−3.18 ± 0.61
0.047
hsa-miR-195
−
−2.70 ± 0.04
0.019
hsa-miR-203
−
−3.23 ± 0.09
0.030
hsa-miR-361-5p
−
−3.56 ± 0.59
0.030
hsa-miR-363
−
−1.66 ± 0.19
0.041
hsa-miR-532-3p
−
−2.92 ± 0.11
0.039
hsa-miR-590-3p
−
−5.20 ± 0.62
0.022
hsa-miR-141
+
3.84 ± 0.22
0.049
hsa-miR-34a
+
5.49 ± 0.18
0.008
hsa-miR-429
−
−3.72 ± 0.89
0.023
miRNA
with mock radiation showed the same. However, miR-302 replacement along with 5 Gy radiation, led to a significant decrease in tumor size. Granting a similar resistance, but opposite of miR-302, another group found miR-95 was being over-expressed by breast cancer cells after ionizing radiation and concluded the resistance was due to miR-95 targeting the sphingolipid phosphatase SGPP1.35 In another study authors found that level of miR-200c expression was linked to the radio-tolerance of breast cancer cells. The radiosensitivity of the cells was achieved by increasing the expression of miR-200c. The authors indicated that the over-expression miR-200c could contribute to inhibition of cell proliferation and increasing apoptosis and DNA breaks, and therefore could be an ideal target for increasing the efficacy of radiation therapy.40
Hormone Therapy With the use of hormone therapy limited to few cancers, the acquired resistance to the treatment after
prolonged usage creates an obstacle to overcome for successful treatments and its continued use. Two hormone treatment resistances discussed here, which were recently linked to the dysregulation of miRNAs in the cells, are Tamoxifen and Fulvestrant resistance. miR-342,41 miR-375,42 and miR-30154 were all found to play a key role in Tamoxifen resistance. When MCF-7 Tamoxifen sensitive and Tamoxifen resistant MCF-7/HER2Δ16 cells were analyzed, a significant downregulation of miR-342 was discovered in the resistant cells. Upon restoring the miR-342 expression, the cell line became sensitized to Tamoxifen-induced apoptosis and showed a dramatic reduction in cell growth.41 Examining different Tamoxifen resistant cells, another group found that miR-375 to be one of the most downregulated miRNAs.42 Re-expression of miR-375 was found to be sufficient to sensitize the cells to Tamoxifen and partially reverse an epithelial–mesenchymal transition (EMT)-like state.42 Switching to Fulvestrant, it was found that miR-221/222 controlled the resistance
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(a)
(b) 350
MDA-MB-231RR/ctrl oligo with 1 Gy irradiation MDA-MB-231RR/ctrl oligo with 5 Gy irradiation
250 200
100 50
MDA-MB-231RR/miR302a with 1 Gy irradiation MDA-MB-231RR/miR302a with 5 Gy irradiation
0 Day
0
2
8
10
12
120 100 80 60 40 20
*
0 R 1R -23 a B 2 M A- -30 MD /miR
Comparison of mRNA levels in xenografts (fold)
(d) Mean tumour weight to ctrll (%)
(c)
6
4
MDA-MB-231RR /ctrl oligo
150
MDA-MB-231RR /miR-302a
Tumour volume (mm3)
300
8
miR-302a *
AKT1
RDA52
6 4 2 0 –2 –4 –6
Control miR-302a
*
*
FIGURE 1 | Effects of miR-302 overexpression in radiosensitivity in vivo. Xenografts in mice derived from miR-302a-transfected MDA-MB-231RR and control vector infected were irradiated, with a strong dosage of 5 Gy or a mock dosage of 1 Gy, to view the efficacy of miR-302a in resensitizing tumors. The cells that were transfected with miR-302a and given high radiation doses showed a drastic decrease in tumor volume, while the controls and mock radiation levels did not. (Reprinted with permission from Ref 36. Copyright 2013 Springer)
inversely of miR-342 and miR-375. The overexpression of miR-221/222 was found in the resistant cell lines and the levels were found to increase after treatment with Fulvestrant in Fulvestrant-sensitive cells. Along with the above, the authors demonstrated that miR-221/222 was essential for the growth and progression of the cells with the acquired resistance.28
Targeted and Other Therapies Despite being a technique that is not employed without an additional treatment, resistance to such a powerful treatment proves problematic as targeted therapy emerges as a standalone cancer therapy. Owing to it being a relatively new treatment, not many studies have been conducted to investigate how resistance to targeted therapy arises. Despite this fact, miR-21 has already been implicated to mediate resistance in breast cancer cells with Trastuzumab treatment which is classified under a targeted therapy.55 One study demonstrated
that miR-21 expression was upregulated and led to Trastuzumab resistance from long-term exposure to the antibody which is cons. Blocking the action of miR-21 was found to re-sensitize the cells to the antibody treatment.55 Interestingly, a different group also found miR-21 to function in immunoresistance, however, they found that inhibition of miR-21 enhanced the release of chemo-attractants in breast cancer cells and increased lymphocyte migration by PIAS3 and STAT3 signaling.56 Another group used a functionalized graphene oxide platform to increase the chemo-sensitivity of breast cancer cell lines to Adriamycin by silencing miR-21. The graphene oxide particles, loaded with anti-sense oligonucleotides and Adriamycin, were used to sensitize the cancer cells to chemotherapy. Subsequent release of the Adriamycin payload induced higher cytotoxicity in breast cancer cells than free Adriamycin.57 Finally, the list of deregulated miRNAs which were found in resistant phenotypes of breast cancer is summarized in Table 3.
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The role of miRNAs in resistance
TABLE 3 List of miRNAs that Play a Role in Resistance to Cancer Therapies Deregulated miRNAs1
Conferred Resistance Doxorubicin
Therapeutic Approach2
References
miR-200b in tumors
miR-200b (up)
44
miR-200c in tumors
miR-200c (up)
44
miR-128 (down)
miR-128 (up)
33
miR-298 (down)
miR-298 (up)
45
miR-326 (down)
miR-326 (up)
32
miR-100 (up)
—
46
miR-29a (up)
miR-29a (down)
46
miR196a (up)
—
46
miR-222 (up)
miR-222 (down)
46
miR-30a (up)
—
46
miR-21 (up)
miR-21 (down)
57 47,48
Paclitaxel
miR-125b (up)
miR-125b (down)
Docetaxel
miR-29a (up)
miR-29a (down)
46
miR-222 (up)
miR-222 (down)
46
miR-34a (up)
miR-34 (down)
49
miR-141 (up)
—
49
miR-7 (down)
—
49
miR-16 (down)
—
49
miR-30a (down)
—
49
miR-125a-p (down)
—
49
miR-126 (down)
—
49
Mitoxantrone
miR-328 (down)
miR-328 (up)
50
Cisplatin
miR-146a
—
43
miR-10a
—
43
miR-221/222
—
43
miR-345
—
43
miR-200b
—
43
miR-200c
—
43
miR-505 (down)
miR-505 (up)
51
miR-21 (up)
miR-21 (down)
34
miR-155 (up)
miR-155 (down)
52
miR-19 (up)
miR-19 (down)
53
miR-302 (down)
miR-302 (up) in xenograft
36
miR-95 (up) in cells and tissue specimens
—
35
miR-200c (down)
miR-200c (up)
40
miR-342 (down)
miR-342 (up)
41
miR-375 (down)
miR-375 (up)
42
miR-301 (up) in tissue specimens
miR-301 (down) in cells and mice
54
Fulvestrant
miR-221/222 (up)
—
28
Trastuzumab
miR-21 (up)
miR-21 (down) in cells and xenograft
55
Immunotherapy
miR-21 (up) in tumors
miR-21 (down)
56
Multidrug
Radiotherapy
Tamoxifen
1 Unless
otherwise stated, deregulated miRNAs were observed in cell lines. Up and down indicates the expression level found in resistant phenotypes. otherwise stated, the therapeutic approach was performed on cell lines. Up and down in this column indicates the regulation of miRNAs experimentally for therapeutic result.
2 Unless
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(b)
cRGD
Cy5.5 Anti-miR10b Cy3
(c) MN-scr-miR
MN-anti-miR10b
8 6 4
p < 0.0001
2 0
MN-scr-miR
MN-anti-miR10b
(d) 160,000 1x105
6x104
2x104 Radiance
Radiance (photons/sec)
MN-anti-miR10b
miR-10b relative expression (SNORD44)
(a)
120,000 80,000 p ≤ 0.01
40,000 0
MN-scrmiR
MN-antimiR10b
FIGURE 2 | Prevention of breast cancer metastasis by targeting miR-10b. (a) A schematic of the nanodrug targeting miRNA-10b (MN-anti-miR10b). The nanodrug consists of magnetic nanoparticles conjugated to Cy5.5 dye, a tumor-targeting peptide (cRGD) and a knock-down LNA oligonucleotide targeting human miRNA-10b. (b) qRT-PCR shows that the nanodrug mediated a significant 87.8 ± 6.2% knock-down of the target miR-10b. (c) Representative bioluminescence images of tumor-bearing mice treated with the anti-miR10b for four weeks beginning prior to lymph node metastasis. The mice were treated either with the active MN-anti-miR10b or with inactive control MN-scr-miR. While the bioluminescence signal was visible in the brachial lymph nodes of control animals, the signals in the experimental animals was at pre-metastatic levels, indicating prevention of metastasis by MN-anti-miR10b. (d) Quantitative analysis of bioluminescence images. Radiance (photons/sec) in the brachial lymph nodes of control mice treated with MN-scr-miR was significantly higher than in experimental animals treated with MN-anti-miR10b. (Reprinted with permission from Ref 17. Copyright 2013 Nature Publishing Group)
miRNAs’ ROLE IN PROLIFERATION AND METASTASIS Many studies have shown miRNAs not only play a role in resistance, but their dysregulation is also key in proliferation of the cancer cells and the promotion of metastasis. As these traits are what we aim to avoid with treatment, and why resistance to them causes complications in the treatment process, it is important to take note of miRNAs that lead to such states. A multitude of different groups have shown proliferation to be altered by changes in miRNAs such as miR-505,51 miR-125b,48 miR-95,35 miR-124,58 and miR-301.54 Increased expression of miR-125b, miR-95, and miR-301 (in different cell lines) was found to give a phenotype of increased proliferation35,54 and, in regard to miR-125b, less apoptosis.48 Opposite of this, increased expression of miR-505 and miR-124 was actually found to exhibit a more positive phenotype with significantly inhibited cell growth.51,58 When looking at metastasis, different authors found the upregulation of miR-10b,24
miR-9,59 miR-22,60 and miR-301a61 in metastatic cell lines. It has been also demonstrated that inhibiting the function of these oncomiRs prevents the spreading of breast cancer to other tissues.15,17 In one recent study, controlling the expression level of miR-10b with locked nucleic acid (LNA) functionalized anti-sense oligonucleotides inhibited the metastasis of human breast cancer cells to lymph nodes in vivo. As seen in Figure 2, the animals with human breast tumor implants do not display metastasis of luciferase expressing cancer cells with anti-miR10b treatment, however, animals with negative treatment with a scrambled oligonucleotide develop metastasis during this time.17
CONCLUSION The role of miRNAs in breast cancer, more specifically their dysregulation, has been shown to be a determining factor in the acquired resistance to treatments in breast cancer cells. With the number of new
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The role of miRNAs in resistance
publications increasing in this field rapidly, it is only a matter of time until the mechanisms of the miRNAs in each of the resistance phenotypes is better understood. Unlike mRNAs, one miRNA can regulate multiple protein expressions. Therefore targeting or controlling the expression of a specific miRNA can potentially alter the expression of multiple proteins. We believe it is important to identify the role of miRNAs in breast cancer therapies to better challenge
the disease. Using miRNA therapeutics to sensitize the breast tumors may potentially decrease the administered amount of anti-cancer drug agents or irradiation necessary in chemotherapy or radiotherapy and in turn, give the patients a better result with treatment. One day it could be possible to combine the breast cancer therapy methods with miRNA therapeutics to fight the disease more effectively while avoiding resistance.
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© 2014 John Wiley & Sons, Ltd.
The role of microRNA in resistance to breast cancer therapy Neil M. Robertson1 and Mehmet V. Yigit2∗ MicroRNAs (miRNAs) are small noncoding RNA molecules with big implications in cancer. The abnormal expression of specific miRNAs has been linked to development of many cancer types. Dysregulated miRNAs play a significant role in proliferation, invasion, differentiation, apoptosis, and resistance of various cancer cells, and considered as oncogenes or tumor-suppressor genes. Findings have shown abnormal expression of specific miRNAs in breast tumors is a strong indication about the resistance to conventional cancer therapy methods. Acquired cancer resistance is a complex, multifactorial occurrence that requires various mechanisms and processes, however, recent studies have suggested that resistance may be linked to treatment-induced dysregulation of miRNAs. This dysregulation of miRNAs can affect the protein expression in cells, the ability for anti-cancer drugs to reach their targets within cells, and the apoptotic pathways. Controlling the expression of these miRNAs alters the resistant phenotype of breast cancer to a nonresistant one. This review focuses on the role of dysregulated miRNAs in breast cancer that are linked to resistance against chemo-, radiation, hormone, and targeted therapies. Finally, the role of miRNAs in breast cancer metastasis is briefly discussed. © 2014 John Wiley & Sons, Ltd. How to cite this article:
WIREs RNA 2014. doi: 10.1002/wrna.1248
INTRODUCTION
M
icroRNAs (miRNAs) are endogenously expressed small noncoding RNA molecules (∼22 nt), which are involved in posttranscriptional regulations of numerous different target messenger RNAs. They bind to the 3′ untranslated region (UTR) of the mRNAs and downregulate target proteins by translational repression or by degradation of mRNAs. miRNAs play a critical role in several stages of various cancer types.1 They are involved in multiple cellular pathways, including cell proliferation, invasion, differentiation, and apoptosis, and act as oncogenes or tumor suppressor genes. The deregulated expression of certain miRNAs in cancer cells has been a strong ∗ Correspondence 1 Department
to: [email protected]
of Chemistry, University at Albany, SUNY, Albany,
NY, USA 2 Department of Chemistry and RNA Institute, University at Albany,
SUNY, Albany, NY, USA Conflict of interest: The authors have declared no conflicts of interest for this article.
indication for different cancer types and the disease stage.2–14 Controlling the expression of these abnormally expressed miRNAs has resulted in successful therapeutic outcomes in cultured cancer cells and animal models.15–17 miRNAs are considered as important biomarkers and therapeutic targets for cancer.18 It has been shown that many dysregulated miRNAs play critical roles in breast cancer, which is the most common cancer among American women and one of the leading causes of cancer related deaths in women worldwide.19–25 According to the American Cancer Society, death rates from breast cancer have been declining for the past two decades due, in part, to improved treatments along with earlier detection and implementation of screening programs. The desire for early detection and screening programs allows for the identification of miRNA signatures associated with different breast tumors, not only for therapeutic options, but also for better diagnosis. While the improved treatments have led to our current methods of chemotherapy, radiation therapy, hormone therapy, and targeted therapy. However, resistance to these
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Advanced Review
therapy methods could develop after a period of time, which decreases the efficacy of the treatment dramatically. It has been demonstrated that abnormal expression of specific miRNAs has strong implications in the resistance against these therapy methods.26–31 Chemotherapy is a treatment which relies on drugs to inhibit the growth and progression of the tumor and to induce cancer cell death. Chemotherapeutic anti-cancer agents (1) can be taken orally or by injection to enter the bloodstream and circulate throughout the body, known as systemic chemotherapy, or (2) can be applied directly into an area for effect, known as regional chemotherapy. The administration depends on the cancer type and stage, however, both are effective means of chemotherapy. Chemotherapy is often used for cancer that has occurred at multiple organs or spread to the other tissues. Chemotherapy is effective, but also carries the problem of side effects due to the death of healthy cells along with cancer cells because of the nonselectivity of the drugs. Another serious problem is the acquisition of drug-resistance in the cancer cells over time. Findings have shown miRNAs to play a key role in drug-resistance, intricately controlling the appearance of the drug-resistant phenotype by their aberrantly expressed levels.32–34 Despite the problems, chemotherapy remains an effective approach in the cancer treatment process. Radiation therapy, radiotherapy, is another mainstay treatment method that uses different types of radiation to inhibit the progression of cancer. With two different types of radiation therapy, external radiation and internal radiation, the choice of therapy is again dependent on the cancer type and stage. Though an effective means of destroying cancer cells, radiation therapy is best used against a single tumor or mass. Much like chemo-resistance, radio-resistance appears in cancer cells after prolonged exposure to the treatment. It has been described that miRNA dysregulation in these cells is, at least in part, responsible for the radio-resistance in the breast tumors.35–40 Hormone therapy is the treatment that blocks or reduces the hormone levels to inhibit the progression of cancer cells. Since some hormones are known to cause certain cancers to grow, blocking these hormones greatly slows the progression of the disease and increases the chance of disappearance of the tumor. While a good treatment, hormone therapy is limited to very few cancers, breast cancer included as one, and is only able to slow the progression. It must be combined with another therapy to induce cancer cell death. Along with the selectivity of the treatment, the acquired resistance to the hormone over time due
to miRNA dysregulation remains problematic in its continued use.41,42 Lastly, targeted therapy is performed using drugs or sometimes small molecules to identify and target tumors without damaging the healthy tissues. Within targeted therapy, multiple techniques including monoclonal antibody therapies and tyrosine kinase inhibitors are found. Monoclonal antibody therapy is administered by infusion and utilizes antibodies to target molecules necessary for the cancer cells to survive. By attaching to the specific molecules, they block the growth rate of the cancer cells, prevent them from spreading, or even induce cancer cell death. Tyrosine kinase inhibitors are utilized similar to block the signals necessary for the malignant cells to grow. While powerful techniques, the therapies have only been approved for use in specific cancer types and are usually applied along with another treatment, such as chemotherapy. Also, a recurring theme is seen with prolonged treatment, the patients develop resistance to it and often another targeted therapy to overcome this resistance is not available. This acquired resistance to targeted therapies is caused, in some cases, by the irregular expression of miRNAs.28 miRNAs can act as oncogenes—oncomiRs—or tumor-suppressor genes by either repressing translation or inducing specific mRNA cleavage.1 The above cancer treatments all share a few traits, one of which being the abnormal expression of miRNAs playing a role in the problems arising from continual use of the treatments. The studies discussed in this review were performed on tumor samples, cultured breast cancer cells or animal models. The list of miRNAs found to play a role, and the effects they bring, is continuously expanding as new reports are published. Their role in other aspects of cancer is also being well studied and there are numerous reports on miRNAs role in breast cancer metastasis and proliferation which is only briefly discussed in this review article.24
miRNAs’ ROLE IN RESISTANCE TO CANCER THERAPY The dysregulation of miRNAs not only plays an important role in cancer development and progression, but has also been found to intricately control the acquisition of drug-resistance and, as a result, the overall success of cancer therapies. Acquired cancer resistance is a complex, multifactorial occurrence that requires various mechanisms and processes, however, recent studies have suggested that chemo-resistance may be linked to drug-induced dysregulation of miRNAs.43 This dysregulation of miRNAs can affect the protein expression in cells,
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The role of miRNAs in resistance
the ability for anti-cancer drugs to reach their targets within cells, and the apoptotic pathways. The past and ever increasing present studies have demonstrated that miRNAs confer resistance to all currently used treatments against breast cancer.
Chemotherapy It has been known that certain proteins [multidrug resistance protein 1 (MDR1)/p-glycoprotein, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP/ABCG2)] play a role in the chemo-resistance of breast cancer cells. As more studies are published, findings are pointing toward the expression of miRNAs being a key regulator of these proteins and their function in the resistance to specific anti-cancer drugs.
Doxorubicin Resistance With doxorubicin (also known as Adriamycin) being a commonly used drug in chemotherapy, the acquired resistance, due to miRNA up- or downregulation, becomes problematic with its continued use in treatment. However, recent studies have shown that altering the levels of the miRNAs in the cells can lead to a promising re-sensitization of the cells. For example, the miRNA-200 family (miR-200b, -200c), specifically the upregulation of them, regulates the transcription of E-cadherin and leads to an overall reversion to a less aggressive cancer phenotype and an increased sensitivity to doxorubicin in resistant cancer cells.44 Similarly, another study demonstrated that the levels of miR-128 in breast tumor-initiating cells to be significantly reduced and accompanied by an over expression of the resistant proteins BMI-1 and ABCC5. Ectopic expression of miR-128 reduced these protein levels, decreased cell viability, and increased cell apoptosis and DNA damage in the presence of doxorubicin.33 Just as increasing the expression of the miR-200 family and miR-128 increased sensitivity to doxorubicin, other studies showed that increased expression of miR-29845 and miR-32632 was found to regulate the doxorubicin chemo-resistance in other cell lines. In cells resistant to doxorubicin, the miR-298 levels were reduced, which lead to an increase in p-glycoprotein and prevented doxorubicin from reaching the nucleus of the cells. The overexpression of miR-298 reversed this effect, with an increased nuclear accumulation of doxorubicin and cytotoxicity observed.45 Likewise, downregulation of miR-326 created a resistant phenotype and when the levels were elevated, the cell lines became sensitized to a doxorubicin treatment.32 Reduced expression levels of miRNAs seem to be a common trend with
doxorubicin resistance in breast cancer cell lines, however, it is not always the case. In MCF-7/Adr resistant cells, five upregulated miRNAs (miR-100, miR-29a, miR-196a, miR-222, and miR-30a) were classified as potential causes for the resistant phenotype. Results showed it was miR-222 and miR-29a that played the largest role, with mimics and inhibitors partially changing the drug-resistance of breast cancer cells.46
Taxane Resistance Other very well known, and commonly used, drugs in the treatment of breast cancer are Paclitaxel (Taxol) and Docetaxel. miRNA dysregulation has recently been found to play a key role in the level of resistance to these core treatment drugs. miR-125b upregulation in taxol-resistant cells caused an inhibition of taxol-induced cytotoxicity and apoptosis.47 This was due to miR-125b suppression of the pro-apoptotic protein Bcl-2 antagonist killer 1 (Bak1), found to be a direct target of the miRNA. Inhibition of miR-125b (or increasing the levels of Bak1) overcame the miRNA-mediated taxol resistance and recovered sensitivity to taxol. Another study showed that expression of miR-125b in vivo led to increased resistance and a more advanced carcinoma in patients, shown in Table 1. miR-125b may be an efficient biomarker to determine effectiveness of treatment and also a suitable target to affect Taxol sensitivity, (Table 1).48 Other miRNAs, miR-29a and miR-222, previously stated to play a role in doxorubicin resistance, were also found be at least partially responsible for acquired resistance to Docetaxel. Mimics and inhibitors of miR-29a and miR-222 were found to alter the drug-resistance of the breast cancer cell line.46 However, another study showed they were not the only miRNAs found to affect Docetaxel resistance.49 The resistance was associated with increased expression of miR-34a and miR-141 and decreased expression of miR-7, miR-16, miR-30a, miR-125a-5p, and miR-126, some of the many miRNAs found dysregulated are organized in Table 2.49 The response to Docetaxel in resistant cells was found to be enhanced with an inhibition of miR-34a.
Mitoxantrone Resistance With Mitoxantrone being used for treatment of metastatic breast cancer, there is no surprise that increasing sensitivity to the drug could improve the survival rate in breast cancer patients. miR-328 was found to negatively regulate the expression of the extremely important breast cancer resistance protein (BCRP/ABCG2) in human cells.50 There is an inverse relationship between miR-328 and BCRP/ABCG2
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TABLE 1 miR-125b Was Found to Be a Key Biomarker for the Success of Treatment in Breast Cancer Patients. (Reprinted with permission from Ref 48. Copyright 2012 Public Library of Science; PLOS) M (SD)
n (total 56)
miR-10b
miR-34a
miR-125b
miR-155
35 (63%)
1.5 (0.7)
3.5 (1.6)
**2.2 (0.4)
*0.9 (0.2)
2.2 (1.1)
5.1 (2.6)
**6.8 (2.1)
*3.1 (0.6)
1.7 (0.6)
3.8 (1.9)
*2.6 (0.7)
1.5 (0.4)
2.1 (1.0)
4.9 (1.2)
*6.1 (1.9)
2.3 (1.0)
1.9 (0.9)
3.9 (1.3)
3.6 (1.1)
1.7 (0.5)
Characteristics
Subgroups
Clinical stage
II III
21 (38%)
Grade
II
32 (57%)
III
24 (43%)
Distant metastasis
M0
20 (36%)
M1
36 (64%)
2.1 (0.9)
4.5 (2.1)
5.9 (1.8)
2.1 (1.2)
Node metastasis
N1-3
40 (71%)
1.5 (0.7)
3.2 (1.1)
*3.1 (0.5)
1.9 (1.0)
N≥4
16 (29%)
2.3 (1.2)
5.2 (2.6)
*6.3 (1.1)
2.1 (0.9)
Negative
13 (23%)
2.1 (1.1)
4.1 (1.9)
4.6 (1.3)
2.2 (1.1)
ER
Positive
43 (77%)
1.9 (0.8)
4.7 (1.9)
4.1 (1.1)
1.9 (0.6)
PR
Negative
19 (34%)
1.9 (0.5)
3.9 (1.5)
3.9 (0.9)
1.6 (0.5)
Positive
37 (66%)
2.2 (1.3)
4.6 (2.1)
4.8 (1.5)
2.3 (1.2)
HER2
Negative
46 (82%)
1.7 (0.8)
3.8 (0.9)
3.8 (1.4)
2.1 (0.9)
Positive
10 (18%)
2.0 (1.2)
4.5 (1.3)
4.2 (1.8)
1.7 (1.1)
ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor. Upregulation of miR-125b led to increased resistance and a more advanced carcinoma, poor characteristics in advanced stages of breast cancer. Patients’ characteristics at the time of primary diagnosis of breast cancer and correlation with serum miRNA levels. *p < 0.05, **p < 0.01.
with miR-328 upregulation resulting in an increased Mitoxantrone sensitivity in MCF-7/MX100 cells.50
Cisplatin Resistance Cisplatin was the first member of a class of platinum-containing anti-cancer drugs and an important chemotherapy drug to trigger apoptosis. In MCF-7 breast cancer cells resistant to cisplatin, a large number of dysregulated miRNAs were discovered. The most significantly dysregulated were miR-146a, miR-10a, miR-221/222, miR-345, miR-200b, and miR-200c while miR-345 and miR-7 were found to target the human multidrug resistance protein 1, MDR1.43
Multidrug Resistance Not all miRNAs seem to affect a specific drug alone or maybe the miRNAs above have not yet been fully explored as to which resistances they confer, regardless, there are many newly found miRNAs that are responsible for a multidrug resistance in human breast cancer cells. For example, a recent study showed that miR-505 inversely correlates to Akt3 and modulates drug sensitivity in the MCF-7-ADR cell line.51 Another study demonstrated that the production of miR-21 lead to a decrease in the tumor-suppressor protein PDCD4, which resulted in the upregulation of inhibitors of apoptosis proteins and MDR1. They used a specific anti-miR-21 inhibitor to silence miR-21
expression and enhance PDCD4 expression, which blocked the mediated cell behavior.34 Other miRNAs to take note of their regulation are miR-155, where the knockdown rendered cells vulnerable to apoptosis and enhanced chemo-sensitivity,52 and miR-19, where inhibition restored sensitivity to cytotoxic agents in multidrug-resistant cells.53
Radiotherapy Radiation therapy is an extremely powerful tool in the treatment of breast cancer and is often highly utilized alongside chemotherapy. The acquired resistance found in breast cancer cells after prolonged exposure has led to problems and in some cases the failure of the treatment. Therefore, the discovery of miRNA dysregulation playing a key role in the radio-resistance was an important step for future clinical work. One miRNA that was found to be abnormally expressed in irradiated breast cancer cells was miR-302.36 The authors found miR-302 to be downregulated and replacement therapy re-sensitized the cells to radiation therapy in vitro and in vivo, see Figure 1.36 In this study, mice xenografts, resistant to radiation, were transfected with miR-302 or controls and then dosed with radiation to kill cells, 5 Gy radiation, or a mock level, 1 Gy radiation. As depicted in Figure 1, the controls remained resistant to radiation and showed no loss in tumor size and miR-302 replacement
© 2014 John Wiley & Sons, Ltd.
WIREs RNA
The role of miRNAs in resistance
TABLE 2 Docetaxel Resistance in Cancer Cell Lines Was Found to be Associated With Increased or Decreased Levels of miRNAs. Results represent mean fold changes based on duplicate experiments. A miRNA expression in docetaxel-resistant cells compared to docetaxel-sensitive cells, where sensistive cells have a fold change of +1. Decreased miRNA expression is indicated with a − (minus); increased miRNA expression is indicated with a + (plus). (Reprinted with permission from Ref 49. Copyright 2012 Springer)
Cell Line MCF-7
MDA-MB-231
miRNA Expressiona
Fold Change (±SD)
P Value
hsa-miR-7
−
−3.87 ± 0.08
0.018
hsa-miR-16
−
−2.64 ± 0.01
0.034
hsa-miR-30a
−
−2.03 ± 0.21
0.010
hsa-miR-30d
−
−2.58 ± 0.32
0.048
has-miR-125a-5p
−
−1.88 ± 0.11
0.042
hsa-miR-126
−
−2.96 ± 0.55
0.025
hsa-miR-129-3p
−
−6.95 ± 4.51
0.006
hsa-miR-138
−
−6.29 ± 0.61
0.045
hsa-miR-149
−
−2.24 ± 0.18
0.023
hsa-miR-151-3p
−
−2.37 ± 0.16
0.007
hsa-miR-151-5p
−
−2.79 ± 0.46
0.009
hsa-miR-190b
−
−3.18 ± 0.61
0.047
hsa-miR-195
−
−2.70 ± 0.04
0.019
hsa-miR-203
−
−3.23 ± 0.09
0.030
hsa-miR-361-5p
−
−3.56 ± 0.59
0.030
hsa-miR-363
−
−1.66 ± 0.19
0.041
hsa-miR-532-3p
−
−2.92 ± 0.11
0.039
hsa-miR-590-3p
−
−5.20 ± 0.62
0.022
hsa-miR-141
+
3.84 ± 0.22
0.049
hsa-miR-34a
+
5.49 ± 0.18
0.008
hsa-miR-429
−
−3.72 ± 0.89
0.023
miRNA
with mock radiation showed the same. However, miR-302 replacement along with 5 Gy radiation, led to a significant decrease in tumor size. Granting a similar resistance, but opposite of miR-302, another group found miR-95 was being over-expressed by breast cancer cells after ionizing radiation and concluded the resistance was due to miR-95 targeting the sphingolipid phosphatase SGPP1.35 In another study authors found that level of miR-200c expression was linked to the radio-tolerance of breast cancer cells. The radiosensitivity of the cells was achieved by increasing the expression of miR-200c. The authors indicated that the over-expression miR-200c could contribute to inhibition of cell proliferation and increasing apoptosis and DNA breaks, and therefore could be an ideal target for increasing the efficacy of radiation therapy.40
Hormone Therapy With the use of hormone therapy limited to few cancers, the acquired resistance to the treatment after
prolonged usage creates an obstacle to overcome for successful treatments and its continued use. Two hormone treatment resistances discussed here, which were recently linked to the dysregulation of miRNAs in the cells, are Tamoxifen and Fulvestrant resistance. miR-342,41 miR-375,42 and miR-30154 were all found to play a key role in Tamoxifen resistance. When MCF-7 Tamoxifen sensitive and Tamoxifen resistant MCF-7/HER2Δ16 cells were analyzed, a significant downregulation of miR-342 was discovered in the resistant cells. Upon restoring the miR-342 expression, the cell line became sensitized to Tamoxifen-induced apoptosis and showed a dramatic reduction in cell growth.41 Examining different Tamoxifen resistant cells, another group found that miR-375 to be one of the most downregulated miRNAs.42 Re-expression of miR-375 was found to be sufficient to sensitize the cells to Tamoxifen and partially reverse an epithelial–mesenchymal transition (EMT)-like state.42 Switching to Fulvestrant, it was found that miR-221/222 controlled the resistance
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Advanced Review
(a)
(b) 350
MDA-MB-231RR/ctrl oligo with 1 Gy irradiation MDA-MB-231RR/ctrl oligo with 5 Gy irradiation
250 200
100 50
MDA-MB-231RR/miR302a with 1 Gy irradiation MDA-MB-231RR/miR302a with 5 Gy irradiation
0 Day
0
2
8
10
12
120 100 80 60 40 20
*
0 R 1R -23 a B 2 M A- -30 MD /miR
Comparison of mRNA levels in xenografts (fold)
(d) Mean tumour weight to ctrll (%)
(c)
6
4
MDA-MB-231RR /ctrl oligo
150
MDA-MB-231RR /miR-302a
Tumour volume (mm3)
300
8
miR-302a *
AKT1
RDA52
6 4 2 0 –2 –4 –6
Control miR-302a
*
*
FIGURE 1 | Effects of miR-302 overexpression in radiosensitivity in vivo. Xenografts in mice derived from miR-302a-transfected MDA-MB-231RR and control vector infected were irradiated, with a strong dosage of 5 Gy or a mock dosage of 1 Gy, to view the efficacy of miR-302a in resensitizing tumors. The cells that were transfected with miR-302a and given high radiation doses showed a drastic decrease in tumor volume, while the controls and mock radiation levels did not. (Reprinted with permission from Ref 36. Copyright 2013 Springer)
inversely of miR-342 and miR-375. The overexpression of miR-221/222 was found in the resistant cell lines and the levels were found to increase after treatment with Fulvestrant in Fulvestrant-sensitive cells. Along with the above, the authors demonstrated that miR-221/222 was essential for the growth and progression of the cells with the acquired resistance.28
Targeted and Other Therapies Despite being a technique that is not employed without an additional treatment, resistance to such a powerful treatment proves problematic as targeted therapy emerges as a standalone cancer therapy. Owing to it being a relatively new treatment, not many studies have been conducted to investigate how resistance to targeted therapy arises. Despite this fact, miR-21 has already been implicated to mediate resistance in breast cancer cells with Trastuzumab treatment which is classified under a targeted therapy.55 One study demonstrated
that miR-21 expression was upregulated and led to Trastuzumab resistance from long-term exposure to the antibody which is cons. Blocking the action of miR-21 was found to re-sensitize the cells to the antibody treatment.55 Interestingly, a different group also found miR-21 to function in immunoresistance, however, they found that inhibition of miR-21 enhanced the release of chemo-attractants in breast cancer cells and increased lymphocyte migration by PIAS3 and STAT3 signaling.56 Another group used a functionalized graphene oxide platform to increase the chemo-sensitivity of breast cancer cell lines to Adriamycin by silencing miR-21. The graphene oxide particles, loaded with anti-sense oligonucleotides and Adriamycin, were used to sensitize the cancer cells to chemotherapy. Subsequent release of the Adriamycin payload induced higher cytotoxicity in breast cancer cells than free Adriamycin.57 Finally, the list of deregulated miRNAs which were found in resistant phenotypes of breast cancer is summarized in Table 3.
© 2014 John Wiley & Sons, Ltd.
WIREs RNA
The role of miRNAs in resistance
TABLE 3 List of miRNAs that Play a Role in Resistance to Cancer Therapies Deregulated miRNAs1
Conferred Resistance Doxorubicin
Therapeutic Approach2
References
miR-200b in tumors
miR-200b (up)
44
miR-200c in tumors
miR-200c (up)
44
miR-128 (down)
miR-128 (up)
33
miR-298 (down)
miR-298 (up)
45
miR-326 (down)
miR-326 (up)
32
miR-100 (up)
—
46
miR-29a (up)
miR-29a (down)
46
miR196a (up)
—
46
miR-222 (up)
miR-222 (down)
46
miR-30a (up)
—
46
miR-21 (up)
miR-21 (down)
57 47,48
Paclitaxel
miR-125b (up)
miR-125b (down)
Docetaxel
miR-29a (up)
miR-29a (down)
46
miR-222 (up)
miR-222 (down)
46
miR-34a (up)
miR-34 (down)
49
miR-141 (up)
—
49
miR-7 (down)
—
49
miR-16 (down)
—
49
miR-30a (down)
—
49
miR-125a-p (down)
—
49
miR-126 (down)
—
49
Mitoxantrone
miR-328 (down)
miR-328 (up)
50
Cisplatin
miR-146a
—
43
miR-10a
—
43
miR-221/222
—
43
miR-345
—
43
miR-200b
—
43
miR-200c
—
43
miR-505 (down)
miR-505 (up)
51
miR-21 (up)
miR-21 (down)
34
miR-155 (up)
miR-155 (down)
52
miR-19 (up)
miR-19 (down)
53
miR-302 (down)
miR-302 (up) in xenograft
36
miR-95 (up) in cells and tissue specimens
—
35
miR-200c (down)
miR-200c (up)
40
miR-342 (down)
miR-342 (up)
41
miR-375 (down)
miR-375 (up)
42
miR-301 (up) in tissue specimens
miR-301 (down) in cells and mice
54
Fulvestrant
miR-221/222 (up)
—
28
Trastuzumab
miR-21 (up)
miR-21 (down) in cells and xenograft
55
Immunotherapy
miR-21 (up) in tumors
miR-21 (down)
56
Multidrug
Radiotherapy
Tamoxifen
1 Unless
otherwise stated, deregulated miRNAs were observed in cell lines. Up and down indicates the expression level found in resistant phenotypes. otherwise stated, the therapeutic approach was performed on cell lines. Up and down in this column indicates the regulation of miRNAs experimentally for therapeutic result.
2 Unless
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Advanced Review
(b)
cRGD
Cy5.5 Anti-miR10b Cy3
(c) MN-scr-miR
MN-anti-miR10b
8 6 4
p < 0.0001
2 0
MN-scr-miR
MN-anti-miR10b
(d) 160,000 1x105
6x104
2x104 Radiance
Radiance (photons/sec)
MN-anti-miR10b
miR-10b relative expression (SNORD44)
(a)
120,000 80,000 p ≤ 0.01
40,000 0
MN-scrmiR
MN-antimiR10b
FIGURE 2 | Prevention of breast cancer metastasis by targeting miR-10b. (a) A schematic of the nanodrug targeting miRNA-10b (MN-anti-miR10b). The nanodrug consists of magnetic nanoparticles conjugated to Cy5.5 dye, a tumor-targeting peptide (cRGD) and a knock-down LNA oligonucleotide targeting human miRNA-10b. (b) qRT-PCR shows that the nanodrug mediated a significant 87.8 ± 6.2% knock-down of the target miR-10b. (c) Representative bioluminescence images of tumor-bearing mice treated with the anti-miR10b for four weeks beginning prior to lymph node metastasis. The mice were treated either with the active MN-anti-miR10b or with inactive control MN-scr-miR. While the bioluminescence signal was visible in the brachial lymph nodes of control animals, the signals in the experimental animals was at pre-metastatic levels, indicating prevention of metastasis by MN-anti-miR10b. (d) Quantitative analysis of bioluminescence images. Radiance (photons/sec) in the brachial lymph nodes of control mice treated with MN-scr-miR was significantly higher than in experimental animals treated with MN-anti-miR10b. (Reprinted with permission from Ref 17. Copyright 2013 Nature Publishing Group)
miRNAs’ ROLE IN PROLIFERATION AND METASTASIS Many studies have shown miRNAs not only play a role in resistance, but their dysregulation is also key in proliferation of the cancer cells and the promotion of metastasis. As these traits are what we aim to avoid with treatment, and why resistance to them causes complications in the treatment process, it is important to take note of miRNAs that lead to such states. A multitude of different groups have shown proliferation to be altered by changes in miRNAs such as miR-505,51 miR-125b,48 miR-95,35 miR-124,58 and miR-301.54 Increased expression of miR-125b, miR-95, and miR-301 (in different cell lines) was found to give a phenotype of increased proliferation35,54 and, in regard to miR-125b, less apoptosis.48 Opposite of this, increased expression of miR-505 and miR-124 was actually found to exhibit a more positive phenotype with significantly inhibited cell growth.51,58 When looking at metastasis, different authors found the upregulation of miR-10b,24
miR-9,59 miR-22,60 and miR-301a61 in metastatic cell lines. It has been also demonstrated that inhibiting the function of these oncomiRs prevents the spreading of breast cancer to other tissues.15,17 In one recent study, controlling the expression level of miR-10b with locked nucleic acid (LNA) functionalized anti-sense oligonucleotides inhibited the metastasis of human breast cancer cells to lymph nodes in vivo. As seen in Figure 2, the animals with human breast tumor implants do not display metastasis of luciferase expressing cancer cells with anti-miR10b treatment, however, animals with negative treatment with a scrambled oligonucleotide develop metastasis during this time.17
CONCLUSION The role of miRNAs in breast cancer, more specifically their dysregulation, has been shown to be a determining factor in the acquired resistance to treatments in breast cancer cells. With the number of new
© 2014 John Wiley & Sons, Ltd.
WIREs RNA
The role of miRNAs in resistance
publications increasing in this field rapidly, it is only a matter of time until the mechanisms of the miRNAs in each of the resistance phenotypes is better understood. Unlike mRNAs, one miRNA can regulate multiple protein expressions. Therefore targeting or controlling the expression of a specific miRNA can potentially alter the expression of multiple proteins. We believe it is important to identify the role of miRNAs in breast cancer therapies to better challenge
the disease. Using miRNA therapeutics to sensitize the breast tumors may potentially decrease the administered amount of anti-cancer drug agents or irradiation necessary in chemotherapy or radiotherapy and in turn, give the patients a better result with treatment. One day it could be possible to combine the breast cancer therapy methods with miRNA therapeutics to fight the disease more effectively while avoiding resistance.
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