It has been proposed that tumor recurrences following treatment with anticancer chemotherapy are driven by a subpopulation of CSCs (26
). Compared with the bulk tumor cell population, CSCs are thought to possess intrinsic resistance to chemotherapy. Indeed, gene expression data from tumors that remain in the breast after neoadjuvant chemotherapy for invasive breast cancer have revealed enrichment of a cancer stem cell-like signature (26
).Consistent with this notion, persistence of tumor cells in the breast after chemotherapy is associated with a high rate of metastatic recurrences and shorter survival compared with that in patients that exhibit a pathological complete response (2
). In an effort to discover targetable signaling pathways present in drug-resistant cells, we examined gene expression signatures enriched by chemotherapy in primary breast cancers. RNA extracted from primary breast tumors before and after chemotherapy showed enrichment of CSC-related genes and a TGF-β–responsive gene signature that was also associated with the more aggressive basal-like subtype of breast cancer. We also examined levels of phosphorylated SMAD2 in tumor cell nuclei by IHC. Staining intensity and percentage of P-SMAD2–positive nuclei were not different in archival tumor sections before and after treatment (data not shown). These results concur with recent findings that a TGF-β–responsive signature was a better predictor of recurrence than P-SMAD2 tumor levels in a large cohort of patients with breast cancer (28
In this study, treatment of TNBC cells with the microtubule inhibitor paclitaxel induced TGF-β reporter activity and upregulated genes in the TGF-β pathway. In some cases this was associated with increased phosphorylation of SMAD2. These data suggest an effect of paclitaxel on TGF-β signaling, which could be direct and/or indirect through negative selection, that is by sparing those CSCs with high TGF-β signaling. However, data in Figure argue for a direct effect, albeit by molecular mechanisms that require further investigation: treatment with the type I TGF-β receptor kinase inhibitor LY2157299 and RNAi-mediated knockdown of SMAD4 inhibited paclitaxel-induced SUM159 and BT549 mammosphere formation and ALDH+ or CD44hi/PROCR+ cells, respectively. Our data do not exclude a simultaneous effect through negative selection. Nonetheless, data in Figure E suggest that might be the case: paclitaxel eliminates ALDH– cells but spares ALDH+ cells. Addition of LY2157299 eliminates paclitaxel-treated ALDH+, suggesting that these cells harbor autocrine TGF-β signaling causally associated with resistance to paclitaxel.
We also showed that paclitaxel induces transcription and secretion of the CSC-associated cytokine IL-8 to drive CSC expansion. Fas ligand and IL-8 are released by tumors upon treatment with chemotherapy (29
). Of note, an IL8
gene expression signature correlates with poor prognosis in patients with basal-like breast cancer (30
), and IL-8 has been shown to expand the CSC fraction in breast cancers (21
). Blockade of IL-8 has also been reported to abrogate chemotherapy-induced enrichment of CSCs (21
). Further, like TGF-β, IL-8 is an osteolytic factor that can facilitate bone metastasis (32
). Both cytokines can expand the CSC population, which may explain the high recurrence rates observed in patients with TNBC. Taken together, these data suggest that following anticancer therapy, upregulation of autocrine/paracrine TGF-β signaling at tumor sites and in situ levels of IL-8 contribute to a tumor microenvironment that sustains CSCs in the primary cancer and also facilitates progression at metastatic sites.
We also showed a causal association between TGF-β signaling and IL-8 expression, since the latter was blocked by treatment with the TGF-βR1 kinase inhibitor LY2157299, a TGF-β type II receptor–neutralizing antibody and SMAD4 siRNA. However, we acknowledge the inconsistency between the efficacy of LY2157299 observed in vitro and in vivo in this article. We would like to point out that the variable monotherapeutic efficacy of TGF-β inhibitors in 2-dimensional and 3-dimensional culture systems has been observed and reported (34
). Phase I safety and dose-finding clinical trials with LY2157299 have shown that it is well tolerated (35
). Efficacy studies using an intermittent schedule of this small molecule are currently ongoing in patients with hepatocellular carcinoma and multiple myeloma (37
). In the xenograft studies shown herein, mouse body weight and histological examination of organs did not reveal any evidence of toxicity related to the TGF-β inhibitor. Further, the combination of LY2157299 with paclitaxel significantly reduced IL-8 expression, stem cell markers, and the tumor-initiating potential of SUM159 cancer cells compared with either drug alone.
The TGF-β type II receptor–neutralizing antibody, TR1 (12
), also blocked chemotherapy-induced expansion of CSCs (Supplemental Figure 9C) by FACS analysis. Since this antibody blocks access of endogenous ligands to TGF-βRII (12
), this result implies that chemotherapy-mediated induction of IL-8 follows autocrine activation of the type II receptor. This is also consistent with previous reports showing that TGF-βRII can activate MAPK (38
) and PI3K (39
) independently of TGF-βR1 and that RNA interference of p38MAPK
abrogates IL-8 expression in a mouse model of MDA231 bone metastasis (40
). Further, TGF-β can still induce p38MAPK-dependent invasion and IL-11 secretion in SMAD4-null MDA468 cells (40
). In data not shown herein, treatment with LY2157299 abrogated paclitaxel-induced enrichment of CSCs in MDA468 cells. Because of the potential pharmacological advantages of antibodies and the high specificity of TR1 to TGF-βRII and the TGF-β pathway, this monoclonal antibody should also be pursued in the clinic in parallel to LY2157299 for the treatment of patients with TNBC.
In summary, results presented herein link anticancer chemotherapy, autocrine TGF-β signaling, IL-8 expression, and the expansion of cells with phenotypic markers of stem cells with tumor-initiating capacity. We surmise that, in the clinic, this phenomenon is associated with metastatic cancer recurrences and poor patient outcome. These data also imply that anticancer chemotherapy in combination with systemic antagonists of TGF-β signaling, either small molecule kinase inhibitors or therapeutic antibodies, should limit basal-like breast tumor recurrences following chemotherapy and, as a result, improve the outcome of patients with this subtype of breast cancer.