**3. The dysregulation of MicroRNAs in breast cancer stem cells**

MicroRNAs (miRNAs) are endogenously synthesized small non-coding RNAs, 19-25 nucleotides in length that negatively regulate gene expression by repressing translation of target mRNAs or targeting them for degradation80. The active miRNA is produced by the RNase III enzyme Dicer in the cytosol from a precursor-miRNA (pre-miRNA) by removing the loop of the pre-miRNA stem-loop. The Dicer-processed miRNA is then taken up by the RNA-induced silencing complex (RISC), which becomes activated when one strand (the antisense or guide strand) is incorporated into the complex and the other strand separates and is discarded. The activated RISC complex can then seek out target mRNAs, which have partially complementary sequences to the guide strand (often in their 3'-UTR), and suppress their translation into protein81.

MiRNA expression is altered in cancer cells and can be used to predict tumor type and prognosis. Cancer-associated miRNAs are frequently deleted, mutated or associated with satellite DNA expansions in cancers, suggesting that these molecules serve as important regulators of tumor development82. Emerging evidence has made it clear that miRNAs also function as important regulators of stemness, collaborating in the maintenance of the pluripotency, control of self-renewal, and differentiation of both normal stem cells and CSCs83. Except for certain miRNAs have high level transcripts, the global downregulation of miRNAs are present in CSCs when compared to their differentiated counterparts82. Dysregulation of miRNAs may result in excessive self-renewal and survival of CSCs which is a likely cause for the chemo-resistance and relapse in tumor patients.

MiRNAs can serve as either tumor suppressors or oncogenes depend on their expression levels in CSCs. Tumor suppressor miRNAs are supposed to inhibit tumor progression while their expression is downregulated. Oncogenic miRNAs are often called oncomiRs and are upregulated in the cancer cells84.

#### **3.1 Tumor suppressors**

Let-7 is the first human miRNA to be discovered and its expression has been observed to be reduced in a number of tumor cell lines including lung and breast cancer85. Recent research indicated let-7 acted as tumor repressor playing an important role in the self-renewal potential of cancer stem cells. Yu and colleagues demonstrated that let-7 family was not expressed by breast CSCs generated from cell lines or 1°patient tumors and increased with differentiation. By expressing of let-7 in breast CSCs or antagonizing let-7 in more

Shown are numbers of 1°, 2°and 3°mammospheres on day 15 from 1000 cells. (D) Mammospheres generated from single-cell cultures of SK-3rd and SKBR3, imaged on indicated day of suspension culture. (E) The majority of freshly isolated SK-3rd cells are CD44+CD24-, while cells with this phenotype are rare in SKBR3. (F) SK-3rd and SKBR3 cells cultured as spheres are CD44+CD24-. When they differentiate in adherent cultures, they gradually assume the parental SBKR3 phenotype, but somewhat more rapidly for SKBR3 mammospheres. (G) When SK-3rd spheres are removed from growth factors, and plated on collagen for 8 hr (top), they do not express luminal (Muc1 and CK-18) or myoepithelial (CK-14 and a-SMA) differentiation markers, while after further differentiation (bottom), they develop into elongated cells with subpopulations staining for either differentiated subtype. (H) Freshly isolated SK-3rd cells are enriched for Hoechstlow SP cells compared with SKBR3

MicroRNAs (miRNAs) are endogenously synthesized small non-coding RNAs, 19-25 nucleotides in length that negatively regulate gene expression by repressing translation of target mRNAs or targeting them for degradation80. The active miRNA is produced by the RNase III enzyme Dicer in the cytosol from a precursor-miRNA (pre-miRNA) by removing the loop of the pre-miRNA stem-loop. The Dicer-processed miRNA is then taken up by the RNA-induced silencing complex (RISC), which becomes activated when one strand (the antisense or guide strand) is incorporated into the complex and the other strand separates and is discarded. The activated RISC complex can then seek out target mRNAs, which have partially complementary sequences to the guide strand (often in their 3'-UTR), and suppress

MiRNA expression is altered in cancer cells and can be used to predict tumor type and prognosis. Cancer-associated miRNAs are frequently deleted, mutated or associated with satellite DNA expansions in cancers, suggesting that these molecules serve as important regulators of tumor development82. Emerging evidence has made it clear that miRNAs also function as important regulators of stemness, collaborating in the maintenance of the pluripotency, control of self-renewal, and differentiation of both normal stem cells and CSCs83. Except for certain miRNAs have high level transcripts, the global downregulation of miRNAs are present in CSCs when compared to their differentiated counterparts82. Dysregulation of miRNAs may result in excessive self-renewal and survival of CSCs which

MiRNAs can serve as either tumor suppressors or oncogenes depend on their expression levels in CSCs. Tumor suppressor miRNAs are supposed to inhibit tumor progression while their expression is downregulated. Oncogenic miRNAs are often called oncomiRs and are

Let-7 is the first human miRNA to be discovered and its expression has been observed to be reduced in a number of tumor cell lines including lung and breast cancer85. Recent research indicated let-7 acted as tumor repressor playing an important role in the self-renewal potential of cancer stem cells. Yu and colleagues demonstrated that let-7 family was not expressed by breast CSCs generated from cell lines or 1°patient tumors and increased with differentiation. By expressing of let-7 in breast CSCs or antagonizing let-7 in more

cells26. Adapted from Yu F, et al.*Cell*, 2007: 131:1109-23.

their translation into protein81.

upregulated in the cancer cells84.

**3.1 Tumor suppressors** 

**3. The dysregulation of MicroRNAs in breast cancer stem cells** 

is a likely cause for the chemo-resistance and relapse in tumor patients.

differentiated cells, it was found that let-7 regulated the key features of breast CSCs—self renewal in vitro, multipotent differentiation, and the ability to form tumors. Because the two targets of let-7 RAS and HMGA2 were responsible for the self renewal and multipotent differentiation, respectively, aberrant expression of let-7 in breast CSCs helps to maintain their stemness26.

Recently, Yu et al. found that similar to let-7, the expression of miR-30 was reduced in breast cancer stem-like cells (BT-ICs), and its target genes, Ubc9, an E2-conjugating enzyme essential for sumoylation, and integrin ß3(ITGB3), were upregulated at protein levels. Overexpression of miR-30 in BT-ICs inhibited their self-renewal ability by repressing Ubc9 and promoted apoptosis by inhibiting Ubc9 and ITGB3. Furthermore, ectopic expression of mir-30 or blocking the expression of Ubc9 in BT-ICs xenografts reduced their tumor-forming capacity and metastasis in NOD/SCID mice, while miR-30 inhibitor enhanced tumorigenesis and metastasis of SKBR3 breast cancer cells with low metastasis potential86. These results suggested that miR-30 could be one of the important miRNAs in regulating the stem-like features of breast cancer

MiR-15/ miR-16 are also tumor suppressors. It was first identified in B cell chronic lymphocytic leukaemia (B-CLL) that miR-15/ miR-16 was lower in their expression level while their target protein the anti-apoptosis Bcl-2 was overexpressed87. The downregulation or deletion of miR-15/miR-16 was also found in other cancer types, such as prostate cancer88, pituitary adenomas89, non-small cell lung cancer (NSCLC)90, and ovarian cancer91. Expression of these miRNAs inhibited cell proliferation, promoted apoptosis, and suppressed tumorigenicity both in vitro and in vivo by targeting multiple oncogenes, including Bcl-2, MCL1, CCND1, Wnt3A and Bmi-1. There has been growing evidence illustrated that the pivotal signaling pathways of the "stem cell genes": Notch, Hedgehog, Wnt, HMGA2, Bcl-2 and Bmi-1 were involved in the self-renewal of CSCs92. Since the oncogenic activation of Bmi-1, Bcl-2 and Wnt3A were frequently correlated with the downregulation of miR-15/miR-16, it was strongly suggested miR-15/miR-16 played a key role in the regulation of CSCs.

MiR-34 has been implicated in cell cycle control related to p5393. In p53 deficicent human gastric cancer cells, restoration of functional miR-34 inhibited the formation of tumorsphere in vitro and tumor initiation in vivo94. In parallel, miR-34 was reported to be involved in pancreatic CSCs self-renewal95. The mechanism of miR-34 mediated suppression of selfrenewal of CSCs was potentially related to the direct modulation of downstream targets Bcl-2 and Notch, suggesting that miR-34 might play an important role in gastric and pancreatic CSCs' self-renewal and/or cell fate determination. However, reduced expression of miR-34a in prostate cancer stem cells facilitated tumor development and metastasis by directly regulating CD44. Accordingly, CD44 knockdown inhibited prostate cancer growth and metastasis96. These results provided a solid experimental basis for developing miR-34a as a promising therapeutic agent against prostate CSCs.

MiR-128 is also a tumor suppressor involved in CSCs. Its expression was dramatically reduced in high grade gliomas, while application of miR-128 inhibited glioma proliferation and self-renewal by targeting Bmi-1 oncogene/stem cell renewal factor97. Same result was found in neural tumor medulloblastoma that miR-128a had growth suppressive activity in medulloblastoma and this activity was partially mediated by targeting Bmi-1 and thereby increasing the steady-state levels of superoxide and promoting cellular senescence. This data has implications for the modulation of redox states in CSCs, which are thought to be resistant to therapy due to their low ROS states98.

Breast Cancer Stem Cells 283

only mesenchymal traits, also cancer stem cell like properties as characterized by their CD44high/CD24low phenotype and increased ability to form mammospheres. On the other hand, HMLE mammospheres expressed markers similar to those of HMLEs that have undergone an EMT77. These findings illustrated EMT cells have cancer stem cell features

MiR-200 is the most discussed family that involved in the regulation of EMT process. Several studies have demonstrated suppression of endogeneous miR-200 family members was sufficient to induce EMT, whereas their ectopic expression induces MET in normal and cancer cell lines through direct targeting of ZEB1/2110. While in CSCs with EMT phenotypes, miR-200 was also detected to be aberrant or absent in breast, pancreas and prostate. Wellner et al showed ZEB1 not only promoted tumor cell dissemination, but also was necessary for the maintaining a stem cell phenotype of pancreatic and colorectal cancer cells by inversely inhibiting the stemness-inhibiting miR-200 family members111. Hence, ZEB/miR-200 feedback loop is a driving force for cancer progression towards metastasis by controlling the state of CSCs. MiR-200 and let-7 both were differentiation associated miRNAs, sometimes they work together regulating the EMT status of CSCs. It has been shown in prostate cancer cells the expression of miR-200 and/or let-7 was decreased in EMT phenotypic tumor cells which also expressed stem-like cell features as defined by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1. Restoration of miR-200 in prostate cancer cells inhibited the EMT process, as well as the clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice which was consistent with the inhibition of Notch1 and Lin28B expression. Along with the decreased expression of Lin28, let-7 was increased which

As discussed above miRNAs are critically involved in the regulation of CSCs and EMT which were considered the "root causes" of chemo-resistant and tumor relapse. Therefore, targeting specific miRNAs could be a very promising therapeutic approach for the treatment optimization aiming at restoring the sensitivity of drug-resistant cells to chemotherapy. If it was possible to introduce miRNA mimics and/or antagonists into CSCs, it could in principle result in reversal of the some of the cells' tumorigenic properties. However, from a clinical/translational research point of view, the critical hurdle to developing this type of approach for cancer therapy is to find an efficient way to selectively deliver miRNAs into CSCs or just cancer cells, but not normal tissues. So far the effective and safe therapeutics are

[1] J, C. Ueber entzundung und eiterung. Path Anat Physiol Klin Med 40, 1-79. (1867). [2] J, C. Congenitales, quergestreiftes Muskelsarkon der Nireren. Virchows Arch, 64-65.

maligni. Arch Memor Observ Chir Pract 11, 217-226. (1874).

bone marrow cells. Radiat Res 14, 213-22 (1961).

into SCID mice. Nature 367, 645-8 (1994).

[3] F, D. Nesso fisio-pathologico tra la struttura dei nei materni e la genesi di alcuni tumori

[4] Till, J. E. & Mc, C. E. A direct measurement of the radiation sensitivity of normal mouse

[5] Lapidot, T. et al. A cell initiating human acute myeloid leukaemia after transplantation

[6] Li, C. et al. Identification of pancreatic cancer stem cells. Cancer Res 67, 1030-7 (2007).

and CSCs exhibit mesenchymal phenotype.

further repressed self-renewal capability112.

still to be studied.

**4. References** 

(1875).

miR-200 is an evolutionary conserved family which were found to be strongly suppressed in CD44+/CD24− lineage human breast cancer cells27 and poorly differentiated pancreatic adenocarcinomas99. Recent research conducted in an inducible oncogenesis model showed that inhibition of miR-200b expression resulted in enrichment of the CSC population, and CSC or mammosphere growth was blocked by overexpression of miR-200b. Meanwhile one of its target Suz12 subunit of PRC2 was increased in CSC which in turn repress the transcription of E-cadherin. Thus, miR-200b acts as a tumor suppressor that blocks the formation and maintenance of mammospheres by targetting Suz12-E-cadherin pathway100. These results identified miR-200 microRNA family as a critical regulator for CSC growth and function.

#### **3.2 Oncogenes**

The miR-17-92 polycistron which is composed of 7 members is found to be overexpressed in multiple tumors, including lung101, lymphoma102, myeloid leukemias103, hepatocellular carcinomas104, medulloblastoma105 and colorectal106. It's known to function as oncogenes to promotes cell proliferation and tumor progression. Introduction of miR-17-92 into hematopoietic stem cells was shown to significantly accelerated the formation of lymphoid malignancies partly by inhibiting apoptosis101. Also Wang et al found members of the miR-17 family were notably more abundant in a mouse model of MLL leukemia stem cells compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Forced expression of miR-17-19b in leukemia cells, was consistent with a higher frequency of leukemia stem cell, reduced differentiation and increased proliferation. The oncogenic effects of miR17-92 on leukemia stem cell self-renewal in MLLassociated leukemia in part due to modulating the expression of p21, a known regulator of normal stem cell function103. Taken together, these studies implicated the miR-17-92 cluster as a potential human oncogene that played a role in cancer stem cells.

The miR-181 has an oncogenic role within cancers as well. MiR-181 family members were up-regulated in EpCAM(+)AFP(+) hepatocellular carcinoma(HCCs) and in EpCAM(+) HCC cells isolated from AFP(+) tumors which have the cancer stem/progenitor cell features. Downregulation of miR-181 reduced EpCAM(+) HCC cell quantity and tumorigenesis, whereas enforced expression of miR-181 in HCC cells resulted in an enrichment of EpCAM(+) HCC cells. The mechamism underlying the regulation of miR-181 on the stemness of EpCAM(+) HCC cells was partially by negatively regulating two hepatic transcriptional regulators of differentiation and an inhibitor of Wnt/\_-catenin signaling (nemo-like kinase [NLK])107. Other evidence also showed miR-181 was elevated in breast cancer stem cells. Overexpression of miR-181a/b, or depletion of its target ataxia telangiectasia mutated(ATM), was sufficient to induce sphere formation in breast cancer cells and promote tumorgenesis108.

#### **3.3 EMT**

The epithelial-mesenchymal transition (EMT) is a vital developmental process that is often activated during cancer invasion and metastasis. During EMT, epithelial cells lose its epithelial characteristics including cell polarity and acquire mesenchymal phenotypes. On the molecular level, cells undergoing EMT down-regulated epithelial markers such as Ecadherin and up-regulated mesenchymal markers such as N-cadherin, vimentin, and fibronectin109. Mani and colleagues were the first group to demonstrated that the immortalized human mammary epithelial cells (HMLEs) undergoing EMT displayed not

miR-200 is an evolutionary conserved family which were found to be strongly suppressed in CD44+/CD24− lineage human breast cancer cells27 and poorly differentiated pancreatic adenocarcinomas99. Recent research conducted in an inducible oncogenesis model showed that inhibition of miR-200b expression resulted in enrichment of the CSC population, and CSC or mammosphere growth was blocked by overexpression of miR-200b. Meanwhile one of its target Suz12 subunit of PRC2 was increased in CSC which in turn repress the transcription of E-cadherin. Thus, miR-200b acts as a tumor suppressor that blocks the formation and maintenance of mammospheres by targetting Suz12-E-cadherin pathway100. These results identified miR-200 microRNA family as a critical regulator for CSC growth

The miR-17-92 polycistron which is composed of 7 members is found to be overexpressed in multiple tumors, including lung101, lymphoma102, myeloid leukemias103, hepatocellular carcinomas104, medulloblastoma105 and colorectal106. It's known to function as oncogenes to promotes cell proliferation and tumor progression. Introduction of miR-17-92 into hematopoietic stem cells was shown to significantly accelerated the formation of lymphoid malignancies partly by inhibiting apoptosis101. Also Wang et al found members of the miR-17 family were notably more abundant in a mouse model of MLL leukemia stem cells compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Forced expression of miR-17-19b in leukemia cells, was consistent with a higher frequency of leukemia stem cell, reduced differentiation and increased proliferation. The oncogenic effects of miR17-92 on leukemia stem cell self-renewal in MLLassociated leukemia in part due to modulating the expression of p21, a known regulator of normal stem cell function103. Taken together, these studies implicated the miR-17-92 cluster

The miR-181 has an oncogenic role within cancers as well. MiR-181 family members were up-regulated in EpCAM(+)AFP(+) hepatocellular carcinoma(HCCs) and in EpCAM(+) HCC cells isolated from AFP(+) tumors which have the cancer stem/progenitor cell features. Downregulation of miR-181 reduced EpCAM(+) HCC cell quantity and tumorigenesis, whereas enforced expression of miR-181 in HCC cells resulted in an enrichment of EpCAM(+) HCC cells. The mechamism underlying the regulation of miR-181 on the stemness of EpCAM(+) HCC cells was partially by negatively regulating two hepatic transcriptional regulators of differentiation and an inhibitor of Wnt/\_-catenin signaling (nemo-like kinase [NLK])107. Other evidence also showed miR-181 was elevated in breast cancer stem cells. Overexpression of miR-181a/b, or depletion of its target ataxia telangiectasia mutated(ATM), was sufficient to induce sphere formation in breast cancer

The epithelial-mesenchymal transition (EMT) is a vital developmental process that is often activated during cancer invasion and metastasis. During EMT, epithelial cells lose its epithelial characteristics including cell polarity and acquire mesenchymal phenotypes. On the molecular level, cells undergoing EMT down-regulated epithelial markers such as Ecadherin and up-regulated mesenchymal markers such as N-cadherin, vimentin, and fibronectin109. Mani and colleagues were the first group to demonstrated that the immortalized human mammary epithelial cells (HMLEs) undergoing EMT displayed not

as a potential human oncogene that played a role in cancer stem cells.

and function.

**3.2 Oncogenes** 

cells and promote tumorgenesis108.

**3.3 EMT** 

only mesenchymal traits, also cancer stem cell like properties as characterized by their CD44high/CD24low phenotype and increased ability to form mammospheres. On the other hand, HMLE mammospheres expressed markers similar to those of HMLEs that have undergone an EMT77. These findings illustrated EMT cells have cancer stem cell features and CSCs exhibit mesenchymal phenotype.

MiR-200 is the most discussed family that involved in the regulation of EMT process. Several studies have demonstrated suppression of endogeneous miR-200 family members was sufficient to induce EMT, whereas their ectopic expression induces MET in normal and cancer cell lines through direct targeting of ZEB1/2110. While in CSCs with EMT phenotypes, miR-200 was also detected to be aberrant or absent in breast, pancreas and prostate. Wellner et al showed ZEB1 not only promoted tumor cell dissemination, but also was necessary for the maintaining a stem cell phenotype of pancreatic and colorectal cancer cells by inversely inhibiting the stemness-inhibiting miR-200 family members111. Hence, ZEB/miR-200 feedback loop is a driving force for cancer progression towards metastasis by controlling the state of CSCs. MiR-200 and let-7 both were differentiation associated miRNAs, sometimes they work together regulating the EMT status of CSCs. It has been shown in prostate cancer cells the expression of miR-200 and/or let-7 was decreased in EMT phenotypic tumor cells which also expressed stem-like cell features as defined by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1. Restoration of miR-200 in prostate cancer cells inhibited the EMT process, as well as the clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice which was consistent with the inhibition of Notch1 and Lin28B expression. Along with the decreased expression of Lin28, let-7 was increased which further repressed self-renewal capability112.

As discussed above miRNAs are critically involved in the regulation of CSCs and EMT which were considered the "root causes" of chemo-resistant and tumor relapse. Therefore, targeting specific miRNAs could be a very promising therapeutic approach for the treatment optimization aiming at restoring the sensitivity of drug-resistant cells to chemotherapy. If it was possible to introduce miRNA mimics and/or antagonists into CSCs, it could in principle result in reversal of the some of the cells' tumorigenic properties. However, from a clinical/translational research point of view, the critical hurdle to developing this type of approach for cancer therapy is to find an efficient way to selectively deliver miRNAs into CSCs or just cancer cells, but not normal tissues. So far the effective and safe therapeutics are still to be studied.
