**4.11 Epithelial to mesenchymal transition and breast cancer stem cells**

An interesting idea that has emerged recently suggests the possibility that cancer cells undergoing EMT acquire stem cell-like characteristics. The breast cancer stem cell (BCSCs) hypothesis contends that breast cancer is derived from a single tumour initiating cell with stem cell-like properties.

BCSCs are characterized as CD24−/low and CD44+ cells which are associated with basal subtype breast cancer. It was first reported by Al-Hajj et al., (2003) when they showed that a CD44+/CD24−/low sub-population of breast cancer cells could produce tumours in a xenograft model more effectively. These cells are regarded as the 'metastatic component' of the cancer, particularly in breast neoplasms as they are the only subset of cells with potential to initiate new tumour growth. This was further supported by analysis of genetic profiles of CD44+ breast cancer cells which showed enrichment with stem-cell markers and displayed activated TGFβ signalling with lung metastasis and poor clinical outcomes (Sheridan et al., 2006; Shipitsin et al., 2007). Furthermore, it has been reported that metaplastic and claudinlow breast cancers are enriched with markers of EMT and display stem cell characteristics suggesting that cancer cells undergoing EMT exhibit stem cell-like characteristics (Prat et al., 2010). In addition to that, inducing EMT in immortalized human mammary epithelial cells with either TGFβ, SNAIL1 and TWIST confers stem cell characteristics with increased formation of mammospheres in three dimensional culture and ductal outgrowths in xenotransplants (Mani et al., 2008; Morel et al, 2008). Interestingly, BCSCs isolated from primary tumors and normal breast tissue showed an increased expression of the mesenchymal markers TWIST1 and 2, FOXC2, SNAIL1, ZEB2, vimentin and fibronectin while epithelial cells (which are CD44-/CD24+) isolated from differentiated carcinoma do not (Mani et al., 2008). Furthermore, hypoxia-induced SNAIL2 expression has also been associated with acquisition of a basal-like breast cancer phenotype with high levels of the stem cell regulatory genes CD133 and BMI1 (Storci et al., 2008). Inhibition of WNT signalling through LRP6 was found to reduce stem cell-like properties and cause EMT reversal, restoration of the epithelial phenotype, and suppression of SNAIL2 and TWIST expression (DiMeo et al., 2009) in a mouse model of breast cancer metastasis to the lung.

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 469

A functionally redundant ER in endocrine-resistant breast cancer might promote a more mesenchymal stem-cell-like phenotype based on the observation that ER negatively regulates the expression of the key EMT transcription factors including SNAIL1 and SNAIL2 (Dhasarathy et al., 2007; Ye et al., 2008). Furthermore, tamoxifen resistant MCF7 cells have been reported to show an enhanced mammosphere formation capacity in comparison to the tamoxifen sensitive cells which suggests an increased CSC fraction (Storci et al., 2008). EMT may facilitate the generation of CSCs with mesenchymal and self-renewal properties necessary for dissemination and initiation of metastasis. (Hollier et al., 2009; Mani et al, 2008). An immunohistochemical analysis of 479 invasive breast carcinomas showed a high expression of the EMT-induced markers vimentin, α-smooth muscle actin, N-cadherin, CDH1, SPARC, laminin and fascin, in comparison to the low expression of E-cadherin in these CD44+/CD24– basal-like breast tumours. These tumours have the ability to form distant metastases hence exhibiting a worse prognosis (Perou et al., 2000; Sorlie et al., 2001). In a study on 117 samples of primary invasive breast carcinomas, nuclear staining of the EMT inducing transcription factor FOXC2 showed a significant correlation with CD44+CD24– basal-like subtypes ( Mani et al., 2007). Another study on 226 blood samples from 39 patients with metastatic breast cancer showed that the majority of the circulating tumour cells (CTCs) exhibited EMT and CSC characteristics (Aktas et al., 2009). CTCs were present in 69 of 226 (31%) blood samples taken from patients with metastatic breast cancer to investigate the expression of TWIST, AKT2, and PI3Kα and ALDH1 which is considered to be a stem cell marker. In the CTC-positive group, 62% were positive for the EMT markers and 69% for ALDH1, while in the CTC-negative group the proportions were 7 and 14%, respectively (Aktas et al., 2009). The CTCs have also been shown to have a reduced expression of epithelial-specific cytokeratins (Pantel et al., 2008). Interestingly, disseminated tumour cells (DTCs) over-expressed TWIST. Assessment of occurrence of bone marrow metastases indicated that TWIST+ cells were present prior to chemotherapy and this was

EMT undergoing CTCs have also been shown to resist apoptosis. One study reported that following the induction of EMT by TGFβ in the EpH-4 and nMuMG murine mammary epithelial cell lines, they tended to acquire resistance to ultraviolet light induced apoptosis (Robson et al., 2006). Likewise, down regulation of the expression of LET-7 miRNA in breast cancer cell lines increased their metastatic potential and the resistance to therapy, in association with the acquisition of stem cell characteristics and EMT-associated gene expression profiles (Yu et al., 2007). Furthermore, the factors that can induce a full EMT; TGFβ, WNT, HEDGEHOG, NOTCH, and RAS signaling pathways, are all considered to be involved in the induction and maintenance of stem cell niches (Fuxe et al., 2010). There is however some data showing that TGFβ stimulation of transformed human breast epithelial cells can result in the loss of stem cell-like properties including the ability to form

It is becoming increasingly apparent that acquired endocrine resistance is a multi-factorial stepwise progression that can be triggered through a number of distinct pathways, that *in vitro*, can be manipulated. Whether it is the actual loss of ER due to transcriptional or translational down-regulation, or functional redundancy of ER (which seems to be the more frequent occurrence *in vivo*), either scenario would have the same end result in terms of

significantly associated with relapse (Watson et al., 2007).

mammospheres (Tang et al., 2007).

**5. Endocrine resistance and EMT** 

It has also been reported that a CD24−/low /CD44+ *in vivo* tumour out- growth which is enriched with EMT markers results from CD8 T-cell-mediated immune response to epithelial breast cancer which would develop characteristics of aggressive carcinomas including potent tumourigenicity, ability to re-establish an epithelial tumour, and enhanced resistance to drugs and radiation (Sheridan et al., 2006; Santisteban et al., 2009). Moreover, breast cancer cells disseminated into the circulation and bone marrow are enriched with CD44+CD24– antigen phenotype (Balic et al., 2006)

EMT induction may be a contributory factor to the decreased efficacy of chemotherapy in breast (Cheng et al., 2007), colorectal (Yang et al., 2006) and ovarian cancer (Kajiyama et al., 2006) while introduction of TWIST into breast cancer cells has been shown to induce paclitaxel resistance. In addition, AKT2 expression, which was amplified in breast cancer has also been correlated with acquired paclitaxel resistance (Cheng et al., 2007). Interestingly, acquisition of enhanced EGFR/ERBB2 signalling in ER+ breast cancer with tamoxifen resistance has been suggested to result from the selection of a more stem cell-like phenotype. EGFR expression is seen in stem cells of the normal mammary gland in both mice and humans (Asselin-Labat et al., 2006; Hebbard et al., 2000) whilst ER is predominantly expressed in the more differentiated luminal cells (Hebbard et al., 2000; Shipitsin et al., 2007). Furthermore, the EGFR pathway is also activated in CSCs of DCIS of the breast and there is emerging evidence for a role of the ERBB2 pathway in the function of CSCs. Expression of ERBB2 and presence of ALDH1+ CSCs was positively correlated in one series of 491 breast cancer patients (Ginestier et al., 2007). The CSC populations of four ERBB2+ breast cancer cell lines have been shown to express more ERBB2 mRNA and protein in comparison to the non-CSC population. Furthermore, trastuzumab was also shown to reduce mammosphere-forming ability and tumourigenicity on serial xenotransplantation (Magnifico et al., 2009). Interestingly, ERBB2+ tumours that received treatment with lapatinib showed decreased EMT related genes in comparison to CD24low/-/CD44+ post treatment tissues from patients that received standard anthracycline-taxane chemotherapy. In addition, the γ secretase inhibitor DAPT or a NOTCH 4 neutralizing antibody significantly reduced mammosphere formation in DCIS. NOTCH pathway antagonism has been reported to enhance the reduction of mammosphere formation in ERBB2 over-expressing cell lines induced by trastuzumab (Magnifico et al, 2009).

Colorectal and lung tumours undergoing EMT display decreased sensitivity to EGFR kinase inhibitors, possibly by the activation of downstream targets PI3K and AKT (Barr et al., 2008). In breast cancer, CD44+/CD24−/low CSCs acquire resistance against the chemotherapeutic agents docetaxel, doxorubicin and cyclophosphamide (Li et al., 2008). Furthermore, a proportion of CD44+/CD24−/low cells increase in breast cancer patients following treatment with these anti-cancer drugs suggesting that breast cancer cells may acquire resistance to both conventional and targeted therapies upon conversion to a mesenchymal-like phenotype. This in turn would suggest that any EMT inducing factors such as TWIST and ERBB2 are crucial players in inducing cancer stem cells.

An analysis of a panel of breast cancer cell lines of luminal, intermediate and basal phenotypes showed a significant increase in the fraction of CSCs (CD44+/CD24low/-/ESA+ ) in basal type breast cancers compared to hormone-sensitive luminal cancers **(**Fillmore & Kuperwasser, 2008). In addition, the number of CSCs and cell line tumourigenicity in *in vivo* models was correlated positively (Fillmore et al., 2008).

It has also been reported that a CD24−/low /CD44+ *in vivo* tumour out- growth which is enriched with EMT markers results from CD8 T-cell-mediated immune response to epithelial breast cancer which would develop characteristics of aggressive carcinomas including potent tumourigenicity, ability to re-establish an epithelial tumour, and enhanced resistance to drugs and radiation (Sheridan et al., 2006; Santisteban et al., 2009). Moreover, breast cancer cells disseminated into the circulation and bone marrow are enriched with

EMT induction may be a contributory factor to the decreased efficacy of chemotherapy in breast (Cheng et al., 2007), colorectal (Yang et al., 2006) and ovarian cancer (Kajiyama et al., 2006) while introduction of TWIST into breast cancer cells has been shown to induce paclitaxel resistance. In addition, AKT2 expression, which was amplified in breast cancer has also been correlated with acquired paclitaxel resistance (Cheng et al., 2007). Interestingly, acquisition of enhanced EGFR/ERBB2 signalling in ER+ breast cancer with tamoxifen resistance has been suggested to result from the selection of a more stem cell-like phenotype. EGFR expression is seen in stem cells of the normal mammary gland in both mice and humans (Asselin-Labat et al., 2006; Hebbard et al., 2000) whilst ER is predominantly expressed in the more differentiated luminal cells (Hebbard et al., 2000; Shipitsin et al., 2007). Furthermore, the EGFR pathway is also activated in CSCs of DCIS of the breast and there is emerging evidence for a role of the ERBB2 pathway in the function of CSCs. Expression of ERBB2 and presence of ALDH1+ CSCs was positively correlated in one series of 491 breast cancer patients (Ginestier et al., 2007). The CSC populations of four ERBB2+ breast cancer cell lines have been shown to express more ERBB2 mRNA and protein in comparison to the non-CSC population. Furthermore, trastuzumab was also shown to reduce mammosphere-forming ability and tumourigenicity on serial xenotransplantation (Magnifico et al., 2009). Interestingly, ERBB2+ tumours that received treatment with lapatinib showed decreased EMT related genes in comparison to CD24low/-/CD44+ post treatment tissues from patients that received standard anthracycline-taxane chemotherapy. In addition, the γ secretase inhibitor DAPT or a NOTCH 4 neutralizing antibody significantly reduced mammosphere formation in DCIS. NOTCH pathway antagonism has been reported to enhance the reduction of mammosphere formation in ERBB2 over-expressing cell lines induced by

Colorectal and lung tumours undergoing EMT display decreased sensitivity to EGFR kinase inhibitors, possibly by the activation of downstream targets PI3K and AKT (Barr et al., 2008). In breast cancer, CD44+/CD24−/low CSCs acquire resistance against the chemotherapeutic agents docetaxel, doxorubicin and cyclophosphamide (Li et al., 2008). Furthermore, a proportion of CD44+/CD24−/low cells increase in breast cancer patients following treatment with these anti-cancer drugs suggesting that breast cancer cells may acquire resistance to both conventional and targeted therapies upon conversion to a mesenchymal-like phenotype. This in turn would suggest that any EMT inducing factors such as TWIST and

An analysis of a panel of breast cancer cell lines of luminal, intermediate and basal phenotypes showed a significant increase in the fraction of CSCs (CD44+/CD24low/-/ESA+ ) in basal type breast cancers compared to hormone-sensitive luminal cancers **(**Fillmore & Kuperwasser, 2008). In addition, the number of CSCs and cell line tumourigenicity in *in vivo*

CD44+CD24– antigen phenotype (Balic et al., 2006)

trastuzumab (Magnifico et al, 2009).

ERBB2 are crucial players in inducing cancer stem cells.

models was correlated positively (Fillmore et al., 2008).

A functionally redundant ER in endocrine-resistant breast cancer might promote a more mesenchymal stem-cell-like phenotype based on the observation that ER negatively regulates the expression of the key EMT transcription factors including SNAIL1 and SNAIL2 (Dhasarathy et al., 2007; Ye et al., 2008). Furthermore, tamoxifen resistant MCF7 cells have been reported to show an enhanced mammosphere formation capacity in comparison to the tamoxifen sensitive cells which suggests an increased CSC fraction (Storci et al., 2008). EMT may facilitate the generation of CSCs with mesenchymal and self-renewal properties necessary for dissemination and initiation of metastasis. (Hollier et al., 2009; Mani et al, 2008). An immunohistochemical analysis of 479 invasive breast carcinomas showed a high expression of the EMT-induced markers vimentin, α-smooth muscle actin, N-cadherin, CDH1, SPARC, laminin and fascin, in comparison to the low expression of E-cadherin in these CD44+/CD24– basal-like breast tumours. These tumours have the ability to form distant metastases hence exhibiting a worse prognosis (Perou et al., 2000; Sorlie et al., 2001). In a study on 117 samples of primary invasive breast carcinomas, nuclear staining of the EMT inducing transcription factor FOXC2 showed a significant correlation with CD44+CD24– basal-like subtypes ( Mani et al., 2007). Another study on 226 blood samples from 39 patients with metastatic breast cancer showed that the majority of the circulating tumour cells (CTCs) exhibited EMT and CSC characteristics (Aktas et al., 2009). CTCs were present in 69 of 226 (31%) blood samples taken from patients with metastatic breast cancer to investigate the expression of TWIST, AKT2, and PI3Kα and ALDH1 which is considered to be a stem cell marker. In the CTC-positive group, 62% were positive for the EMT markers and 69% for ALDH1, while in the CTC-negative group the proportions were 7 and 14%, respectively (Aktas et al., 2009). The CTCs have also been shown to have a reduced expression of epithelial-specific cytokeratins (Pantel et al., 2008). Interestingly, disseminated tumour cells (DTCs) over-expressed TWIST. Assessment of occurrence of bone marrow metastases indicated that TWIST+ cells were present prior to chemotherapy and this was significantly associated with relapse (Watson et al., 2007).

EMT undergoing CTCs have also been shown to resist apoptosis. One study reported that following the induction of EMT by TGFβ in the EpH-4 and nMuMG murine mammary epithelial cell lines, they tended to acquire resistance to ultraviolet light induced apoptosis (Robson et al., 2006). Likewise, down regulation of the expression of LET-7 miRNA in breast cancer cell lines increased their metastatic potential and the resistance to therapy, in association with the acquisition of stem cell characteristics and EMT-associated gene expression profiles (Yu et al., 2007). Furthermore, the factors that can induce a full EMT; TGFβ, WNT, HEDGEHOG, NOTCH, and RAS signaling pathways, are all considered to be involved in the induction and maintenance of stem cell niches (Fuxe et al., 2010). There is however some data showing that TGFβ stimulation of transformed human breast epithelial cells can result in the loss of stem cell-like properties including the ability to form mammospheres (Tang et al., 2007).
