**4.7 HOX genes**

Another important set of genes in regulating EMT is the homeobox (HOX) gene family, master players in regulating embryonic development and maintaining homeostasis through strictly regulated expression in various tissues and organs during adult life. Several studies have demonstrated the association of HOX genes in the pathogenesis of multiple cancers. For example, HOXA7 and HOXD13 have been associated with lung cancer (Lechner et al., 2001), HOXC4 and HOXC8 in prostate cancer (Miller et al., 2003), HOXB7 in ovarian cancer (Naora et al, 2001) and HOXA10 in endometrial cancer (Yoshida et al., 2006). In one study 60% of their breast cancers had no HOXA5 expression (Raman et al., 2000) which causes p53-dependent apoptosis. HOXA5 was reported to cause cell death through the activation of the caspase pathways in HS578T cells expressing mutant p53 (Chen et al., 2004). HOXD10 was extensively reduced as malignancy increased in epithelial cells, and restoring its expression in MDA-MB-231 could significantly reduce the migration capacity of these highly aggressive cells (Carrio et al., 2005). HOXB13 over-expression was associated with increased MCF10A cell motility and invasion *in vitro*, while its ratio to interleukin-17β receptor was predictive of tumour recurrence during adjuvant tamoxifen monotherapy. HOXB7 is involved in tissue remodeling of the normal mammary gland (Ma et al., 2004) and is expressed at higher levels in metastatic breast tumours (Care et al., 1998, 2001). Furthermore, regulation of the expression of several growth and angiogenic factors, including basic FGF, VEGF, IL8, ANG1, ANG2, and MMP9 in SKBR3 breast cancer cells, depends on the over-expressed levels of HOXB7 which can result in the formation of vascularised tumours when grown as xenografts in nude mice. HOXB9 like HOXB7 can lead to increased cell motility and EMT (Hayashida et al., 2010).

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 467

internal ribosomal entry site sequence and thereby increasing the translational efficiency of ZEB2 which then directly inhibits E-cadherin expression, maintaining an EMT state (Beltran

Although these RNA molecules are associated with the regulation of EMT and MET, other miRNAs such as miR-10b are reportedly associated with metastasis and invasion. It inhibits HOX10 translation while increasing RHOC when induced by TWIST (Ma et al., 2007). Another miRNA that seems to increase the metastatic potential of cancer cells is miR-29a; up-regulated in a mesenchymal metastatic RASXT mammary cell line compared to epithelial EpRas cells. In addition, over-expression of miR-29a suppresses expression of tristetraprolin, a regulator of epithelial polarity and metastasis, and leads to EMT and metastasis through RAS signalling. This correlates with data from breast cancer patients showing enhanced miR-29a and reduced tristetraprolin levels (Gebeshuber et al., 2009). In contrast to miR-10b, miR-335 was found to be a suppressor of invasion and metastasis through modulation of the expression of the 'six gene signature' set: COL1A1, MERTK, PLCB1, PTPRN2, TNC and SOX4 which are considered predictive markers of metastasis and invasion. miR-335 was also reported to suppress invasion and metastasis in MDAMB231, a

highly metastatic and invasive ER-ve breast cancer cell line (Tavazoie et al., 2008).

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

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

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.

/CD24+) isolated from differentiated carcinoma do

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

et al., 2008).

stem cell-like properties.

while epithelial cells (which are CD44-
