Enhanced effect on reoccurrence of triple negative breast cancer using TGF-B inhibition combined with chemotherapy
Abstract
Introduction
Breast cancer is the most common type of cancer and breast cancer is causing the second most victims of all types of cancer. The American Cancer Society estimated that in the United States there were 235030 new cases of breast cancer in 2014, in the same year 40430 people suffering breast cancer died (Siegel et al., 2014). Breast cancer is predominantly occurring in women and only sporadic in men. Several factors have been determined as risk factors for developing breast cancer such as aging, obesity, and long exposure with oestrogen. Genetic mutations in genes coding for BRCA or the Human Epidermal growth factor Receptor 2 (HER-2/ErbB-2) have also been determined as risk factors for breast cancer (Kaminska et al., 2015). Three major types of breast cancer are characterized, HR positive, HER2 positive and triple negative. HR-positive cancer overexpresses estrogen receptors (ER) and progesterone receptors (PR) and can be treated with several hormone therapies. HER2 positive breast cancer is negative for ER and PR, but it is HER2 positive and therefore treatable with anti-HER2 drugs. Accounting for 15% of the cases triple negative breast cancers (TNBC) do not overexpress the HER2 receptor and lacks detectable ER and PR. An alternative therapy against TNBC is required because regular hormone therapies are not helpful (Tang et al., 2016)
Tumour heterogeneity, for example caused by epidermal growth factor (EGF), is cancer cell diversity within a tumour and has an important effect on the prognosis, response to therapies, and metastasis of breast cancer. The different types of cancer cells in the same tumour are clonally related but show a distinct genetic expression profile. These expression profiles cause activation of different signalling pathways (Shipitsin et al., 2007). Many therapies still fall short resulting in reduced overall survival of breast cancer patients. Tumour heterogeneity could explain why therapies are failing to treat breast cancer. The tumour’s functioning as a complex ecosystem may enhance tumour fitness (Juntilla et al., 2013). Breast cancer therapies might be improved when there is additional information about heterogeneously expressed growth factors. Breast cancer cell subpopulation analysis showed some tumour cells are
more tumour genic while other tumour cells are not tumour genic. Cancer Stem Cells (CSCs) are an example of tumour genic cells that can self-renew and give rise to tumours that display phenotypic heterogeneity with their parental tumours (Kreso et al., 2014).
Tumor Growth Factor B (TGF-B) is such a heterogeneously expressed growth factor, which is providing cells with developmental programmes and it controls cell behaviour (Massagué, 2012). High TGF-B levels are correlated with poor prognosis in breast cancer due to the fact that TGF-B has a profound effect on tumour progression through modification of cell behaviour of carcinoma cells (Bierie et al., 2010). The poor prognosis of breast cancer is intriguing because the TGF-B pathway can promote cell growth arrest, which should not be collaborative for tumour progression (Siegel et al., 2003). Malfunctioning of TGF-B can result in severe diseases because signalling by TGF-B will promote an invasive phenotype and the acquisition of mesenchymal characteristics in breast cancer cells (Leivonen et al., 2007, Lesko e., et al.). TGF-B alone can induce epithelial-to-mesenchymal transition (EMT) of breast cancer cells, after EMT breast cancer cells acquire the potential of mesenchymal cells including motility and invasiveness (Moustakas et al., 2007). Furthermore TGF-B is known to inhibit anti-tumour effects of immune cells, including T-cells, NK cells, neutrophils, monocytes, and macrophages (Li et al., 2006).
In this paper the role of TGF-B in cancer heterogeneity will be reviewed, furthermore it will be studied whether TGF-B is a possible target for an anti-cancer therapy. First an overview of the role of TGF-B in tumour progression will be made. Second studies using intravital imaging TGF-B signalling will be described. At last anti-cancer therapies targeting TGF-B will be compared with each other. The aim is to determine whether TGF-B could be used in anti-cancer therapies and in which way this could be done.
Overview of TGF-B signalling
TGF-B has been widely studied over the last decades and is an important protein in the regulation of tumour progression. In cancer, TGF-B1 was shown to be more abundant than TGF-B2 or TGF-B3. The TGF-B1 levels can be elevated via several mechanisms including expression of αvβ6integrin, calpain, cathepsin D, chymase, elastase, endoglycosidase F, kallikrein, MMP-9, neuraminidase, plasmin, and thrombospondin-1 (TSP-1) (Bierie et al., 2010) Once TGF-B levels are elevated, it will bind to TGF-B serine/threonine kinase receptors that activate the intracellular signal transduction pathways through hetero-tetramerization. Pathways activated by TGF-B are shown in. Downstream signalling of TGF-B consists of phosphorylation of the SMAD dependent pathways, which includes several transcription factors inhibiting cell proliferation, promoting EMT, and induce apoptosis of carcinoma cells. (Bierie et al., 2010) Phosphorylation of SMAD2 and SMAD3 makes them able to form complexes with SMAD4, a common mediator SMAD. (Bierie et al., 2010) Furthermore TGF-B is capable to activate SMAD1 and SMAD5 in epithelial cells, epithelial derived tumour cells and fibroblasts. Together with the SMAD2-SMAD4 and SMAD3-SMAD4 complexes SMAD1 and SMAD5 are important responses to stimulation by the TGF-B ligands. (Daly et al., 2008)
Figure 1: TGF-B signalling pathway. TGF-B ligand binds TGF-B receptor II and drives hetero-tetramerization with TGF-B receptor I. Phosphorylation of R-SMADS by TGF-B receptors activates the canonical signalling. There are several pathways including the SMAD-pathways and the non-canonical (non-SMAD) signalling pathways, which both translocate into the nucleus to bind gene promoters and activate gene expression. (Connolly et al., 2012)
Poor prognosis in breast cancer correlated with high TGF-B levels as described in the introduction, but in paradox mutations or loss of type 1 or type 2 TGF-B receptor, thus low level TGF-B signalling, is as well often correlated with inducing most types of cancer, including breast cancer. (Bieri et al., 2006) TGF-B can promote growth arrest as well as tumour cell motility, but in some cases this paradox is resolved as TGF-B is only promoting tumour cell motility in these cases due to loss of key mediators of the growth inhibiting responses. (Giampieri et al., 2010) next paragraph will be described how TGF-B is able to promote tumour cell motility and what the consequences are of this induced tumour cell motility.
TGF-B promotes single tumour cell motility
Using multiple imaging techniques Giampieri et al. (2009) demonstrated the effects of TGF-B signalling in breast cancer. Rat mammary carcinoma cells (MTLn3E) were used to determine whether signalling promoting cancer cell motility was locally and transiently activated. A TGF-B dependent promoter was utilized (CAGA12-luciferase) to confirm that MTLn3E cells could respond to TGF-B in vivo. Luminescence analyse of the TGF-B treated MTLn3E cells indicated MTLn3E tumours revealed heterogeneous phosphorylation of the SMAD3 protein, which is proof for activation of TGF-B signalling. Tumour staining of MTLn3E and human breast carcinoma showed considerable heterogeneity in TGF-B signalling, phosphorylated SMAD3 was particularly present in margins of the MTLn3E tumour samples. These data confirm TGF-B is signalling is not uniformly active in tumours.
Giampieri et al. (2009) also visualized the nuclear localisation of SMAD2 protein using modified MTLn3E cells expressing GFP-SMAD2 and Orange-NLS to visualise the nucleus. Timelapse analysis of intravital imaging displayed GFP-SMAD2 localisation was heterogeneous in vivo. All cells displaying GFP-SMAD2 in the nucleus were single moving cells. (Figure 2i, 2ii, 2iii) The data Giampieri et al. (2009) presented shows the mode of migration used in vivo by cancer cells correlates with TGF-B signalling.
Figure 2: Accumulation of GFP-SMAD2 in MTLn3E cells.
Intravital timelapse of GFP-SMAD2 and NLS-Orange expressing primary tumours. 2i shows singly moving cells, 2ii cohesive movind cells and 2iii shown non-motile cells. (Giampieri et al., 2009)
Lack of transforming growth factor promotes collective cancer cell invasion. Matise et al. (2012) acquired mammary carcinoma cells from either TGF-B receptor II knockout mice or wildtype mice. The combined fluorescently labelled mammary carcinoma cells with or without a TGF-B receptor II knockout and mammary fibroblast were xenografted onto a chicken embryo chorioallantoic membrane. Intravital microscopy of the xenografts identified two types of phenotype single cell migration or cohesive motility. Mammary carcinoma cells with a full length TGF-B receptor II migrated on their own while mammary carcinoma cells with a TGF-B receptor II knockout moved collectively. TGF-B signalling drives single cell motility and is therefore a target for anti-cancer therapies as single carcinoma cells can form more metastasis. (Figure 3)
No data of the number of metastasis sites was obtained which could be interesting because single moving cells should be able to form more metastasis.
Figure 3: Single cell and collective cell invasive metastatic potentials. A metastasis assay of TGF-B receptor knockout cells and TGF-B full length epithelial cells. TGF-B KO epithelial cells were more able to cohesively extravasate and form greater metastasis. (Matise et al., 2012)
Therapies using TGF-B inhibition
An alternative for TNBC is needed, as most regular therapies are not effective against it. TGF-B is a promising candidate, because TGF-B induces EMT and has been associated with acquisition of tumour stem-like properties. As Shipitsin et al. (2007) discovered TGF-B kinase inhibitors reversed EMT in mammary epithelial cells expressing CD44. In TNBC TGF-B levels are often elevated, suggesting that the TGF-B pathway is involved in maintenance of cancer stem-like cells (CSCs) (Bierie et al., 2008) Distant relapse of breast cancer after chemotherapy is an important clinical issue of TNBC due to the factor of the chemotherapy resistant CSCs. Hollier et al., 2009)
To test whether TGF-B inhibition might be effective against maintenance of CSCs, Bhola et al. (2013) treated xenografted TNBC cells in mice with a microtubule inhibitor paclitaxel, induced TGF-B reporter activity, and upregulated genes in the TGF-B pathway. In some cases treatment with paclitaxel and induced TGF-B receptor activity was associated with increased SMAD2, suggesting an effect of paclitaxel on TGF-B signalling promoting chemotherapy resistance. When a TGF-B receptor I kinase inhibitor (LY2157299) combined with paclitaxel as treatment on mice with xenografted TNBC cells, paclitaxel resistant CSCs were reduced. The same decrease of paclitaxel resistant CSCs was shown using a neutralizing TGF-B type II receptor antibody. This data implies combining chemotherapy with TGF-B kinase inhibitors or therapeutic antibodies limits basal-like breast cancer reoccurrence via CSCs and so enhances chemotherapy action against TNBC.
Park et al. (2015) recently obtained the same chemotherapy enhanced effect of TGF-B inhibition. The effect of TGF-B inhibitor EW-7197 on metastasis was examined xenotransplantating mice with MDA-MB-231 tumour cells. The inoculated mice were treated with nothing, just paclitaxel, just EW-7197 or both paclitaxel and EW-7197. Though just treating the mice with paclitaxel reduced the cancer burden efficiently, the combinatorial therapy of both EW-7197 and paclitaxel significantly decreased lung metastasis as shown in figure 4 by H&E staining.
Figure 4: Analysing lung metastasis with phase-contrast microscopy images of H&E stained lungs. The red triangles mark metastasis sites. Compared to the control group (PAC – and EW -) paclitaxel did not have a significant effect on lung metastasis. Only paclitaxel or only EW-7179 as well had no effect on lung metastasis. Lung metastasis decreased after treatment with paclitaxel and EW7179. (PAC=paclitaxel, EW=EW-7179) (Park et al., 2015)
Described therapies of Bhola et al. and Park et al. combined chemotherapy with either TGF-B receptor antibodies or TGF-B receptor kinase inhibitors. Both studies were done to determine short-term effects (5-6 days). Long term dosing strategies with LY2157299 for TGF-B inhibition may be ill advised. For example, two Drug-related dose limiting toxicities of LY2157299 in patients with grade IV glioma were determined by Rodon et al. (2011). Although there was no significant toxicity, pulmonary embolism and thrombocytopenia were observed. These effects could be significant toxic on the long term. Furthermore TGF-B signalling is involved in many natural physiological functions, so TGF-B may still lead to harmful off-target effects. With this concern in mind, effects of TGF-B inhibition should still be investigated, as it is a very promising strategy to enhance chemotherapy effects.
Box 1 Intravital Imaging techniques
Common techniques such as immunohistochemistry have provided insights in experimental mouse tumour models at discrete time-intervals. Intravital microscopy (IVM) techniques have been developed to reveal both spatial and temporal information. Intravital imaging by Giampieri et al. (2009) revealed temporal information of accumulation of GFP-SMAD2 at different sites shown in figure 2. Visualisation at cellular resolution is often done using two-photon laser scanning microscopy. Fluorophores in tumourcells were excited using two low-energy IR photons. Fluorescence was emitted after excitement in the visible range. IVM studies visualize the interaction between tumour cells and their microenvironment in very high resolution. Potentially types of IVM can be utilized to find more about interaction between tumour cells and their microenvironment and should therefore be used more.
Discussion
TGF-B is an important signalling protein in breast cancer. It can promote growth arrest as well as tumour cell motility, but in some tumours the ability of growth arrest via TGF-B is lost due to malfunctioning of key mediators in the growth arrest pathway. Gampieri et al. (2009) and Matise et al. (2012) determined TGF-B signalling stimulated single cell motility, which is known to induce the number of metastasis sites. When chemotherapy was combined with a TGF-B receptor kinase inhibitor or TGF-B receptor binding antibodies, the effect of the chemotherapy on reoccurrence of breast cancer or metastasis was enhanced.
In conclusion TGF-B might not be a target for anti-cancer therapies against breast cancer, but Bhola et al. and Park et al. TGF-B inhibition is capable of enhancing anti-cancer therapies.
TNBC is not treatable as well as HR positive or HER2 positive types of breast cancer are. Often TNBC reoccurs at a distant site of the primary tumour due to metastasis. TGF-B induces EMT and has been associated with acquisition of tumour stem-like properties leading to metastasis. A chemotherapy enhanced with TGF-B inhibition is a possible solution as treatment of breast cancer in TNBC.
TGF-B can be inhibited in several ways including TGF-B receptor kinase inhibitors and TGF-B receptor antibodies, but it is not clear which way is most effective and has least side effects. Furthermore the combination of chemotherapy and TGF-B inhibition has to be investigated.
Early treating breast cancer with TGF-B inhibition could possibly even prevent metastasis. Results combining chemotherapy with TGF-B inhibition is not only promising for breast cancer treatments but also in several other types of cancer.
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