Abstract
The spread of tumors is a complex process involving a series of sequential steps, such as metastatic subclone cell detachment and vascular invasion, transport and survival in the circulation, arrest at new location, extravasation into the surrounding parenchyma and establishment of new growth. These steps are mediated by genetic changes. Although many metastasis-associated and -suppressor genes have been identified, how cancer cells acquire metastatic abilities is still an open question. In this study, the group led by Terumi Kohwi-Shigematsu found that special AT-rich sequence-binding protein 1 (SATB1) plays a crucial role in determining the metastasis of breast cancer cells [1].
SATB1 is a tissue-specific matrix association region (MAR)-binding protein that participates in the chromatin higher structure package and tissue-specific gene expression. It belongs to a class of transcriptional regulators that function as a ‘landing platform’ for several chromatin-remodeling enzymes and, hence, regulates large chromatin domains [2]. In doing so, it is able to regulate the expression of more than 1000 genes [1,3,4].
SATB1 was first identified as a factor that bound to the nuclear MAR 3’ of the immunoglobulin heavy-chain intronic enhancer in 1992 [5]. Its expression is most abundant in the thymus, with low levels present in the testes, fetal brain and osteoblasts, and virtually undetectable levels found in other tissues [3,5]. SATB1 is already known to play a vital role in the development and maturation of T cells. Thymocyte development is blocked at the CD4+CD8+ double-positive stage in SATB1-null mice [3].
Breast cancer is a leading cause of cancer death in women worldwide. Although the treatment of breast cancer has made significant progress, and survival from breast cancer is improving, distant metastases remain the most common cause of breast cancer recurrence, resulting in more than 410,000 deaths/year worldwide [6]. Several breast cancer metastasis-associated genes have been identified owing to the increased expression in breast cancer metastases [7]. Among the important genes regulated by SATB1, many are already known to play a role in aggressive breast cancers, including the cell-adhesion molecule, human secreted protein acidic and rich in cysteine (SPARC), anti-apoptosis protein B-cell lymphoma 2 (BCL2), an EGF receptor-ligand amphiregulin and IGF-binding protein 2 (IGFBP2) [4,8].
Results
In the paper described here, the authors examined the expression of SATB1 mRNA and protein in 24 breast epithelial cell lines, including normal human epithelial cells, immortalized cells and both nonmetastatic and metastatic breast cancer cells, and found that no SATB1 was detected in normal and immortalized cells, SATB1 expression was detected in cancer cells and high SATB1 expression was only present in metastatic breast cancer cells.
In the clinical setting, they examined 28 human primary breast tumors, and found that SATB1 was detected in all 16 poorly differentiated infiltrating ductal carcinomas (p < 0.0001). Low-level SATB1 expression was found in some moderately differentiated tumor samples (in seven out of 12 samples), and was not present in adjacent nontumor tissue. SATB1 expression was not only restricted to late clinical stages of disease, but was also observed in a subset of primary breast tumors at early clinical stages before lymph-node metastasis occurred.
Using tissue microarrays, the group analyzed 2197 human primary breast cancer tissue samples for which clinical follow-up studies were available. The highest levels of SATB1 were found in samples from patients whose survival times had been shortest; patients whose tumor samples had no SATB1 expression generally had longer survival times (p < 0.001), suggesting that a high level of SATB1 expression is an excellent independent indicator of poor prognosis – independent of whether breast cancer cells have already metastasized to the lymph nodes at the time of diagnosis.
Furthermore, they investigated the effects of downregulating SATB1 by RNA interference on biological behavior of the highly metastatic human breast cancer cell line, MDA-MB-231, and found that this dramatically reduced the invasive capacity of these cells and also reduced their capacity of anchorage-independent growth. These in vitro results were confirmed in vivo. The cells of SATB1 downregulation failed to develop into metastatic tumors after they were injected into the tails of test mice, whereas MDA-MB-231 cells formed metastatic tumors in the lungs once they were injected into mice. By contrast, when a SATB1-expression construct was introduced into the nonmetastatic human breast cancer cell line, SKBR3, it enhanced the malignant behavior of SKBR3 cells. When the ectopically expressing SATB1 cells were injected into the mammary glands of mice, they grew more aggressively compared with the control SKBR3 cells. These ectopically expressing SATB1 cells were also able to form metastatic tumors in the lungs after they were intravenously injected into mice.
In the study investigating the gene-expression profile of MDA-MB-231 cells using either control shRNA or SATB1 shRNA1 (one of two different SATB1 sequences), the authors found that SATB1 globally changed the expression of hundreds of genes. It increases the expression of genes that promote tumor growth and metastasis, and reduces the expression of tumor suppressors, from growth factors to cell signaling to cell-cycle regulation. They are all favorable factors for growth, invasion and metastasis. Among the upregulated genes, many genes are already known to play a role in aggressive breast cancers, such as EGF-receptor protein (erbB2), S100A4 (metastasin), matrix metalloproteinase (MMP)2, 3 and 9, TGF-β1 and TGF-α. SATB1 also represses the expression of the metastasis-suppressor genes breast cancer metastasis suppressor 1 (BRMS1), KAI1/CD82, KISS1 and nonmetastatic 23 (NM23).
Consistent with the authors' observations that SATB1 depletion from MDA-MB-231 cells restored normal cell morphology, SATB1 downregulation blocks the upregulation of cell-structure genes typically observed in invasive breast cancers, suggesting that SATB1 expression also affects the growth of breast epithelial cells.
Using the urea–chromatin immunoprecipitation (ChIP) assay followed by quantitative PCR (qPCR; urea–ChIP–qPCR), the authors studied the mechanisms by which SATB1 regulates gene expression. They found that SATB1 directly regulates some gene expression by influencing the promoter activity, including erbB2, S100A4, Abelson murine leukemia viral oncogene homolog 1 (abl1), TGF-β1, MMP3, laminA/C (LMNA), BRMS1, claudin 1 (CLDN1) and β-catenin (CTNNB1), and also indirectly influences gene expression by recruiting histonemodifying factors to promoters, including glyceraldehtde-3-phosphate dehydrogenase (GAPDH), integrin-β5 (ITBG5) and tissue inhibitor of metalloproteinase 1 (TIMP1).
Significance of the results
At present, no metastasis-specific genes have been identified [9]. The discovery of the role of SATB1 in aggressive breast cancer has fundamental implications for prognosis and possible new treatments for cancers. The role of SATB1 in breast cancer is a new paradigm for other tumors. According to the previous description, cancer cells develop into metastatic cells that need to acquire several abilities in order to overcome various physiological barriers. They are controlled by the various metastasis suppressors.
This finding shows that a protein can control the expression of many genes in the cells. It might be related to the manner by which SATB1 is regulated. Since SATB1 is a MAR-binding protein, MARs have been shown to exhibit widespread transcriptional regulatory functions, whereby they may either promote or inhibit transcription, depending on the context [10]. SATB1 appears to be particularly important in cells that must change their function – nonmetastatic cells develop into metastatic cells as the thymocytes develop into T cells.
If SATB1 expression is mainly found only in aggressive cancer cells and not in nonmetastatic tumor, then this can be very beneficial for doctors. Doctors can classify the tumor subtype, predict risk of metastasis and relapse, assess prognosis and select optimal therapeutic options, according to the expression of SATB1 in cancer cells. Studies strongly suggest that therapies which significantly reduce the risk of distant metastases are likely to improve long-term outcomes.
However, it is still worth using caution when considering the significance of this finding. Although it appears that the expression of SATB1 is acquired as the breast cancer cells become more aggressive, it is possible to indentify other genes in a similar way. For example, silencing of osteopontin (OPN) abrogates tumorigenicity and metastasis of human metastatic breast carcinoma cell line, MDA-MB-435 [11], and similar results are also found in MDA-MB-231 cells [12], suggesting that OPN plays a greater role in breast cancer growth and metastasis. In addition, silencing of ezrin reverses the metastatic behaviors of MDA-MB-231 cells [13]. Although the groups provide no evidence that the ectopically espressing OPN or ezrin may contribute to tumor progression and metastatic tendencies in the nonmetastatic breast cancer cells, I believe this to be true. Moreover, a general view is that, in most cancers, the metastasic ability of cancer cells is already implanted in a small part of cells relatively early in carcinogenesis [9]. It is clinically observable that some tumors spread to distant sites in the body even when primary tumors are still very small. The authors indicate that SATB1 is also found in a subset of primary breast tumors at early clinical stages before lymph-node metastasis occurs [1].
Since SATB1 is a global gene regulator that regulates more than 2–10% of the total genes in the human genome [3,14], and studies in recent years have revealed that it plays major roles in T-cell development, early erythroid differentiation, homeostasis and response to physiological stimuli [3,14–16], this raises questions concerning the specificity of SATB1 as a target in the treatment of human cancers.
Future perspective
So far, the research has been limited to mice. Therefore, a priority must be placed on whether these results can be confirmed in human studies. Although, at present, SATB1 appears to be a master regulator, in order to decide whether breast cancer will spread, it will be important to know whether these results can be reproduced in different laboratories; whether the findings can be confirmed in different types of human cancers; and whether the high SATB1 expression is always identified in metastatic tumors since telomerase – a key regulator for cell immortalization – is only found in malignant rather than in benign tumors. At the same time, several basic questions remain to be answered:
What factor determines the SATB1 expression in the cells?
What other factors may work together with SATB1?
Since metastatic cancer cells are similar to stem cells in many aspects, including cell proliferation, migration and regulation [17,18], it appears plausible to examine the expression of SATB1 in stem cells. A recent report demonstrated that gene-expression profiles in high-grade astrocytomas resembled the profiles in neural stem cells, and that these genes exhibited similar up- or down-expression patterns and were actively involved in cell proliferation, adhesion, migration and metastasis [19].
Besides SATB1, the role of the other MAR-binding proteins, such as scaffold/matrix associated region 1 (SMAR1) and Cux/CDP in cancer metastasis, remains to be further investigated in the future.
Breast cancer is a leading cause of cancer death in women worldwide and distant metastases remain the most common cause of breast cancer.
Special AT-rich sequence-binding protein 1 (SATB1), a special global gene regulator, is found to be highly expressed in aggressive breast cancer cells.
Ectopically expressing SATB1 cells acquire metastatic activity and growth advantage, but SATB1-depleted cells abolish tumorigenesis in mice, suggesting that SATB1 plays a linchpin role in breast tumor growth and metastasis in some circumstances.
Whether SATB1 is also a master regulator for metastasis of other human cancer types remains to be proven.
Metastasis is a selective advantage of tumor cells in tumorigenesis; in fact, the so-called metastasis-controlling genes may not exist in the tumor.
Footnotes
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.