Mechanisms of Endocrine Resistance in Lobular Breast Cancer
The development and progression of breast cancer are affected by many factors, most of which involve a change in expression of certain genes. Of the mechanisms for regulating gene expression, ncRNAs have proven to be integral to the cell in recent studies. ncRNAs include microRNA (miRNA), long noncoding RNA (lncRNA), transfer RNA, ribosomal RNA, and small nucleolar RNA. Two forms of ncRNA that are especially important for regulation of gene expression in cells are miRNA and lncRNA.
miRNAs are small RNA molecules of 18 to 22 base pairs that regulate the expression of target mRNAs by inhibiting translation or degrading the transcripts. After miRNA genes are initially transcribed by RNA polymerase II, a process regulated by transcription factors and nuclear receptors, the transcripts undergo significant processing both in the nucleus and cytoplasm (Figure 2). First, the pri-miRNA sequence is cleaved in the nucleus by Drosha, a class 2 RNase III enzyme that works in conjunction with DiGeorge syndrome chromosomal region 8 (DGCR8). The resulting pre-miRNA hairpin is exported from the nucleus to the cytoplasm via Exportin 5 where it is further cleaved by Dicer, another member of the RNase III family, resulting in a short double-strand piece of RNA. The two strands of the RNA then separate into the passenger strand, which often gets degraded, and the mature miRNA strand, which binds to argonaute (Ago) proteins to form the RNA-induced silencing complex. The miRNA of the RNA-induced silencing complex guides the complex to a complementary mRNA 3′ untranslated region. Complementarity of the miRNA seed sequence (base pairs 2 to 7) to the target mRNA stimulates Ago2 in the RNA-induced silencing complex to degrade the mRNA. However, if there is only partial complementarity, the translation of the mRNA will be inhibited.
(Enlarge Image)
Figure 2.
Standard pathway by which microRNAs are processed and loaded onto RISC to regulate gene expression. Regulation of microRNA (miRNA) expression is controlled at the miRNA promoter by transcription factors (TF) and nuclear receptors (NR). After transcription, the pri-miRNA is processed inside the nucleus by Drosha and DGCR8 to form pre-miRNA – a hairpin miRNA. Exportin 5 exports the pre-miRNA from the nucleus into the cytoplasm where it gets cleaved further by Dicer, resulting in a short double-strand piece of RNA. These strands are separated into the passenger strand, which often gets degraded, and the mature strand, which is loaded onto RNA-induced silencing complex (RISC) for action on target mRNA. If the seed sequence (base pairs 2 to 7) of the mature miRNA is complementary to the mRNA, the transcript is degraded. However, if there is not perfect complementarity between the miRNA seed sequence and its target mRNA, the result is inhibition of translation. DGCR8, DiGeorge syndrome chromosomal region 8.
Expression profiles of miRNA in breast tumor samples have been correlated with biopathologic features such as hormone receptor status and proliferation index, and are used to distinguish between basal and luminal subtypes. For example, miRNAs overexpressed in basal, ERα-negative primary breast cancers include miR-150, which has been shown to promote breast cancer growth, and miR-135b, which correlates with early metastasis of breast cancer cells. miRNAs overexpressed in luminal, ERα-positive breast cancers include miR-126 and miR-10a, which are associated with an increase in patients' relapse-free time after tamoxifen treatment.
Beyond the use of expression profiles, specific miRNAs have been associated with regulation of genes involved in breast cancer. The let-7 miRNA family has been shown to regulate the self-renewal capacity of breast tumor-initiating cells derived from cell lines and primary patient tumors by inhibition of HRAS and high mobility group AT-hook2 (HMGA2) – genes involved in self-renewal and differentiation, respectively. miR-21, which targets phosphatase and tensin homolog (PTEN), was identified to be upregulated in primary patient samples of invasive breast cancer compared with normal breast tissue by miRNA in situ hybridization staining. miR-373 and miR-520c are considered metastasis-promoting miRNAs and are shown to be upregulated in lymph node metastases compared with primary tumor samples. Promotion of tumor invasion and metastasis by miR-373 and miR-520c is probably achieved via suppression of the CD44 gene, which codes for a hyaluronan receptor and has been identified as a metastasis suppressor in breast cancer.
For a more comprehensive look at the miRNAs involved in breast cancer, the reader should refer to reviews by O'Day and Lal and by Singh and Mo.
lncRNAs are ncRNA transcripts longer than 200 base pairs that are transcribed from various genomic locations, such as in the promoters, enhancers, introns, or anti-sense coding regions of genes, or in their own stand-alone position in the genome (Figure 3A). Unlike miRNAs whose primary role is to repress the translation of their mRNA targets, lncRNAs have been shown to act as protein–DNA or protein–protein scaffolds, miRNA sponges, and protein decoys, as well as regulators of translation (Figure 3B). While previously thought of as junk DNA, lncRNAs are now regarded as an important part of the cell's gene regulation machinery, controlling cell cycle, apoptosis, and differentiation.
(Enlarge Image)
Figure 3.
Location of long noncoding RNAs in the genome and roles of long noncoding RNAs in regulation of cellular processes. (A) Long noncoding RNA (lncRNA) genes reside in various genomic locations, such as in the promoters, enhancers, introns, or anti-sense coding regions of genes, and can also be in their own stand-alone position in the genome. These lncRNA genes sometimes contain small RNA genes, like microRNA (miRNA), that are spliced out of the lncRNA after transcription. (B) The actions of lncRNAs affect many cellular processes. lncRNAs may serve as scaffolds to bring nuclear receptors in contact with promoters of their target genes via chromatin looping, or they may recruit an epigenetic modifier to the chromatin. They can also bind proteins, such as transcription factors, to prevent their binding to DNA – similar to their mechanism of miRNA inhibition. Among the effects lncRNAs have on mRNA, translational activation and maintenance of mRNA stability are also important.
Some investigators have begun to identify lncRNAs whose expression is associated with aberrant signaling or unregulated survival of cancer cells. According to the lncRNADisease Database, there are 16 lncRNAs known to play a role in breast cancer including H19, growth arrest-specific 5 (GAS5), homeobox antisense intergenic RNA (HOTAIR), and breast cancer anti-estrogen resistance 4 (BCAR4).
The lncRNA H19, present on the maternal allele, normally plays a role in imprinted regions of the genome to silence insulin-like growth factor 2 (IGF2). H19 was said to have an oncogenic role in breast cancer cells in 2002, but has since been found to exhibit tumor suppressive action in vivo. Hormonal regulation of H19 is important, as estradiol is able to stimulate H19 transcription in MCF-7 breast cancer cells, while tamoxifen inhibits this transcription.
GAS5 expression is downregulated in breast cancer samples relative to adjacent unaffected normal breast tissue, and has a distinct tumor suppressive role in breast cancer by inducing apoptosis and suppressing cell proliferation. GAS5 acts primarily by preventing the glucocorticoid receptor from binding target DNA. Despite additional interactions with the androgen receptor and progesterone receptor, results show that GAS5 does not bind the ER.
HOTAIR was first identified as an lncRNA that regulates the homeobox D (HOXD) cluster by tethering the polycomb repressor complex 2 (PRC2) protein to the DNA at this site. PRC2 is able to promote histone H3K27 trimethylation and subsequent repression of transcription at the HOXD cluster, thereby preventing differentiation and leading to an invasive cellular phenotype. Overexpression of HOTAIR has been associated with enhanced metastasis and invasion of breast cancer cells and can be used as a predictor of overall survival and progression-free survival. A potential explanation for HOTAIR overexpression in breast cancer could be related to the presence of estrogen response elements in the HOTAIR promoter, leading to estradiol-induced HOTAIR expression.
Of the known lncRNAs associated with breast cancer, BCAR4 is most noteworthy for its role in endocrine resistance (see Long noncoding RNAs and tamoxifen resistance).
Noncoding RNAs and Breast Cancer
The development and progression of breast cancer are affected by many factors, most of which involve a change in expression of certain genes. Of the mechanisms for regulating gene expression, ncRNAs have proven to be integral to the cell in recent studies. ncRNAs include microRNA (miRNA), long noncoding RNA (lncRNA), transfer RNA, ribosomal RNA, and small nucleolar RNA. Two forms of ncRNA that are especially important for regulation of gene expression in cells are miRNA and lncRNA.
microRNAs and Breast Cancer
miRNAs are small RNA molecules of 18 to 22 base pairs that regulate the expression of target mRNAs by inhibiting translation or degrading the transcripts. After miRNA genes are initially transcribed by RNA polymerase II, a process regulated by transcription factors and nuclear receptors, the transcripts undergo significant processing both in the nucleus and cytoplasm (Figure 2). First, the pri-miRNA sequence is cleaved in the nucleus by Drosha, a class 2 RNase III enzyme that works in conjunction with DiGeorge syndrome chromosomal region 8 (DGCR8). The resulting pre-miRNA hairpin is exported from the nucleus to the cytoplasm via Exportin 5 where it is further cleaved by Dicer, another member of the RNase III family, resulting in a short double-strand piece of RNA. The two strands of the RNA then separate into the passenger strand, which often gets degraded, and the mature miRNA strand, which binds to argonaute (Ago) proteins to form the RNA-induced silencing complex. The miRNA of the RNA-induced silencing complex guides the complex to a complementary mRNA 3′ untranslated region. Complementarity of the miRNA seed sequence (base pairs 2 to 7) to the target mRNA stimulates Ago2 in the RNA-induced silencing complex to degrade the mRNA. However, if there is only partial complementarity, the translation of the mRNA will be inhibited.
(Enlarge Image)
Figure 2.
Standard pathway by which microRNAs are processed and loaded onto RISC to regulate gene expression. Regulation of microRNA (miRNA) expression is controlled at the miRNA promoter by transcription factors (TF) and nuclear receptors (NR). After transcription, the pri-miRNA is processed inside the nucleus by Drosha and DGCR8 to form pre-miRNA – a hairpin miRNA. Exportin 5 exports the pre-miRNA from the nucleus into the cytoplasm where it gets cleaved further by Dicer, resulting in a short double-strand piece of RNA. These strands are separated into the passenger strand, which often gets degraded, and the mature strand, which is loaded onto RNA-induced silencing complex (RISC) for action on target mRNA. If the seed sequence (base pairs 2 to 7) of the mature miRNA is complementary to the mRNA, the transcript is degraded. However, if there is not perfect complementarity between the miRNA seed sequence and its target mRNA, the result is inhibition of translation. DGCR8, DiGeorge syndrome chromosomal region 8.
Expression profiles of miRNA in breast tumor samples have been correlated with biopathologic features such as hormone receptor status and proliferation index, and are used to distinguish between basal and luminal subtypes. For example, miRNAs overexpressed in basal, ERα-negative primary breast cancers include miR-150, which has been shown to promote breast cancer growth, and miR-135b, which correlates with early metastasis of breast cancer cells. miRNAs overexpressed in luminal, ERα-positive breast cancers include miR-126 and miR-10a, which are associated with an increase in patients' relapse-free time after tamoxifen treatment.
Beyond the use of expression profiles, specific miRNAs have been associated with regulation of genes involved in breast cancer. The let-7 miRNA family has been shown to regulate the self-renewal capacity of breast tumor-initiating cells derived from cell lines and primary patient tumors by inhibition of HRAS and high mobility group AT-hook2 (HMGA2) – genes involved in self-renewal and differentiation, respectively. miR-21, which targets phosphatase and tensin homolog (PTEN), was identified to be upregulated in primary patient samples of invasive breast cancer compared with normal breast tissue by miRNA in situ hybridization staining. miR-373 and miR-520c are considered metastasis-promoting miRNAs and are shown to be upregulated in lymph node metastases compared with primary tumor samples. Promotion of tumor invasion and metastasis by miR-373 and miR-520c is probably achieved via suppression of the CD44 gene, which codes for a hyaluronan receptor and has been identified as a metastasis suppressor in breast cancer.
For a more comprehensive look at the miRNAs involved in breast cancer, the reader should refer to reviews by O'Day and Lal and by Singh and Mo.
Long Noncoding RNAs and Breast Cancer
lncRNAs are ncRNA transcripts longer than 200 base pairs that are transcribed from various genomic locations, such as in the promoters, enhancers, introns, or anti-sense coding regions of genes, or in their own stand-alone position in the genome (Figure 3A). Unlike miRNAs whose primary role is to repress the translation of their mRNA targets, lncRNAs have been shown to act as protein–DNA or protein–protein scaffolds, miRNA sponges, and protein decoys, as well as regulators of translation (Figure 3B). While previously thought of as junk DNA, lncRNAs are now regarded as an important part of the cell's gene regulation machinery, controlling cell cycle, apoptosis, and differentiation.
(Enlarge Image)
Figure 3.
Location of long noncoding RNAs in the genome and roles of long noncoding RNAs in regulation of cellular processes. (A) Long noncoding RNA (lncRNA) genes reside in various genomic locations, such as in the promoters, enhancers, introns, or anti-sense coding regions of genes, and can also be in their own stand-alone position in the genome. These lncRNA genes sometimes contain small RNA genes, like microRNA (miRNA), that are spliced out of the lncRNA after transcription. (B) The actions of lncRNAs affect many cellular processes. lncRNAs may serve as scaffolds to bring nuclear receptors in contact with promoters of their target genes via chromatin looping, or they may recruit an epigenetic modifier to the chromatin. They can also bind proteins, such as transcription factors, to prevent their binding to DNA – similar to their mechanism of miRNA inhibition. Among the effects lncRNAs have on mRNA, translational activation and maintenance of mRNA stability are also important.
Some investigators have begun to identify lncRNAs whose expression is associated with aberrant signaling or unregulated survival of cancer cells. According to the lncRNADisease Database, there are 16 lncRNAs known to play a role in breast cancer including H19, growth arrest-specific 5 (GAS5), homeobox antisense intergenic RNA (HOTAIR), and breast cancer anti-estrogen resistance 4 (BCAR4).
The lncRNA H19, present on the maternal allele, normally plays a role in imprinted regions of the genome to silence insulin-like growth factor 2 (IGF2). H19 was said to have an oncogenic role in breast cancer cells in 2002, but has since been found to exhibit tumor suppressive action in vivo. Hormonal regulation of H19 is important, as estradiol is able to stimulate H19 transcription in MCF-7 breast cancer cells, while tamoxifen inhibits this transcription.
GAS5 expression is downregulated in breast cancer samples relative to adjacent unaffected normal breast tissue, and has a distinct tumor suppressive role in breast cancer by inducing apoptosis and suppressing cell proliferation. GAS5 acts primarily by preventing the glucocorticoid receptor from binding target DNA. Despite additional interactions with the androgen receptor and progesterone receptor, results show that GAS5 does not bind the ER.
HOTAIR was first identified as an lncRNA that regulates the homeobox D (HOXD) cluster by tethering the polycomb repressor complex 2 (PRC2) protein to the DNA at this site. PRC2 is able to promote histone H3K27 trimethylation and subsequent repression of transcription at the HOXD cluster, thereby preventing differentiation and leading to an invasive cellular phenotype. Overexpression of HOTAIR has been associated with enhanced metastasis and invasion of breast cancer cells and can be used as a predictor of overall survival and progression-free survival. A potential explanation for HOTAIR overexpression in breast cancer could be related to the presence of estrogen response elements in the HOTAIR promoter, leading to estradiol-induced HOTAIR expression.
Of the known lncRNAs associated with breast cancer, BCAR4 is most noteworthy for its role in endocrine resistance (see Long noncoding RNAs and tamoxifen resistance).
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