A CAF-Fueled TIMP-1/CD63/ITGB1/STAT3 Feedback Loop Promotes Migration and Growth of Breast Cancer Cells.

TIMP-1 is one of the many factors that CAFs have been shown to secret. TIMP-1 can act in a tumor-supportive or tumor-suppressive manner. The purpose of this study was to elucidate the role of CAF-secreted TIMP-1 for the effects of CAFs on breast cancer cell behavior. Breast cancer cells were exposed to conditioned medium collected from TIMP-1-secreting CAFs (CAF-CM), and the specific effects of TIMP-1 on protein expression, migration and growth were examined using TIMP-1-specifc siRNA (siTIMP1), recombinant TIMP-1 protein (rhTIMP-1) and TIMP-1 level-rising phorbol ester. We observed that TIMP-1 increased the expression of its binding partner CD63 and induced STAT3 and ERK1/2 activation by cooperating with CD63 and integrin ß1. Since TIMP-1 expression was found to be dependent on STAT3, TIMP-1 activated its own expression, resulting in a TIMP-1/CD63/integrin ß1/STAT3 feedback loop. IL-6, a classical STAT3 activator, further fueled this loop. Knock-down of each component of the feedback loop prevented the CAF-induced increase in migratory activity and inhibited cellular growth in adherent cultures in the presence and absence of the anti-estrogen fulvestrant. These data show that TIMP-1/CD63/integrin ß1/STAT3 plays a role in the effects of CAFs on breast cancer cell behavior.

Biological effects and regulation of IGFBP5 in breast cancer.

The insulin-like growth factor receptor (IGF1R) pathway plays an important role in cancer progression. In breast cancer, the IGF1R pathway is linked to estrogen-dependent signaling. Regulation of IGF1R activity is complex and involves the actions of its ligands IGF1 and IGF2 and those of IGF-binding proteins (IGFBPs). Six IGFBPs are known that share the ability to form complexes with the IGFs, by which they control the bioavailability of these ligands. Besides, each of the IGFBPs have specific features. In this review, the focus lies on the biological effects and regulation of IGFBP5 in breast cancer. In breast cancer, estrogen is a critical regulator of IGFBP5 transcription. It exerts its effect through an intergenic enhancer loop that is part of the chromosomal breast cancer susceptibility region 2q35. The biological effects of IGFBP5 depend upon the cellular context. By inhibiting or promoting IGF1R signaling, IGFBP5 can either act as a tumor suppressor or promoter. Additionally, IGFBP5 possesses IGF-independent activities, which contribute to the complexity by which IGFBP5 interferes with cancer cell behavior.

BCL3 expression is strongly associated with the occurrence of breast cancer relapse under tamoxifen treatment in a retrospective cohort study.

Patients with estrogen receptor positive breast cancer are usually receiving an anti-estrogen therapy by either aromatase inhibitors or selective estrogen receptor mediators such as tamoxifen. Nevertheless, acquired resistance to tamoxifen under treatment frequently hampers therapy. One proposed explanation for this phenomenon is the interaction of the tumor cells with cells of the tumor microenvironment via the Insulin-like growth factor RNA binding protein 5/B-cell lymphoma 3 (IGFBP5/BCL3) axis. Here we investigated whether a high expression of BCL3 either cytoplasmic or nuclear is associated with the occurrence of a relapse under anti-estrogen therapy in patients. Formaldehyde-fixed, paraffin-embedded samples of 180 breast cancer patients were analyzed for BCL3 expression by immunohistochemistry. An immunoreactive score (IRS) was calculated from staining intensity in cytoplasm and nucleus as well as the percentage of positive tumor cells. These scores were correlated with clinico-pathological parameters using cross-tabulation analysis and patients' relapse free and overall survival by Kaplan-Meier analysis and Cox regression. A tamoxifen-adapted MCF-7 derived cell line was investigated for BCL3 localization by immunofluorescence. The cytosolic BCL3-IRS significantly correlated with the proliferation marker Ki-67, and with the occurrence of a relapse under tamoxifen treatment. Nuclear score correlated only with tamoxifen-relapse. In survival analysis, both scores were highly significant prognostic factors for relapse free, but not for overall survival. This was especially obvious for estrogen receptor positive and HER2/NEU negative cases as well as lobular breast cancer. Tamoxifen-treated, but not aromatase-treated patients had a poor survival when BCL3 scores were high. A tamoxifen adapted cell line exhibited a reduced expression and mainly nuclear localization of BCL3, compared to the parental estrogen receptor positive cell-line MCF-7. Altogether, these data strongly support a function of BCL3 in tamoxifen resistance and its potential use as a predictive biomarker for tamoxifen resistance.

Nuclear Mechanisms Involved in Endocrine Resistance.

Endocrine therapy is a standard treatment offered to patients with ERα (estrogen receptor α)-positive breast cancer. In endocrine therapy, ERα is either directly targeted by anti-estrogens or indirectly by aromatase inhibitors which cause estrogen deficiency. Resistance to these drugs (endocrine resistance) compromises the efficiency of this treatment and requires additional measures. Endocrine resistance is often caused by deregulation of the PI3K/AKT/mTOR pathway and/or cyclin-dependent kinase 4 and 6 activities allowing inhibitors of these factors to be used clinically to counteract endocrine resistance. The nuclear mechanisms involved in endocrine resistance are beginning to emerge. Exploring these mechanisms may reveal additional druggable targets, which could help to further improve patients' outcome in an endocrine resistance setting. This review intends to summarize our current knowledge on the nuclear mechanisms linked to endocrine resistance.

Carcinoma-Associated Fibroblasts Promote Growth of Sox2-Expressing Breast Cancer Cells.

CAFs (Carcinoma-associated fibroblasts) play an important role in cancer progression. For instance, they promote resistance to anti-estrogens, such as fulvestrant. Here, we show that, in ERα-positive breast cancer cell lines, the cocktail of factors secreted by CAFs (CAF-CM) induce the expression of the embryonal stem cell transcription factor Sox2 (sex determining region Y (SRY)-box 2). Long-term exposure to CAF-CM was able to give rise to very high Sox2 levels both in the absence and presence of fulvestrant. IL-6 (interleukin-6), a major component of CAF-CM, failed to raise Sox2 expression. In MCF-7 sublines established in the presence of CAF-CM, almost all cells showed Sox2 expression, whereas long-term treatment of T47D cells with CAF-CM resulted in a ~60-fold increase in the proportions of two distinct populations of Sox2 high and low expresser cells. Exposure of BT474 cells to CAF-CM raised the fraction of Sox2 high expresser cells by ~3-fold. Cell sorting based on CD44 and CD24 expression or ALDH (aldehyde dehydrogenase) activity revealed that most Sox2 high expresser cells were not CD44hi/CD24lo- or ALDH-positive cells suggesting that they were not CSCs (cancer stem cells), though CD44 played a role in Sox2 expression. Functionally, Sox2 was found to protect CAF-CM-treated cells against apoptosis and to allow higher growth activity in the presence of fulvestrant. Mechanistically, the key drivers of Sox2 expression was found to be STAT3 (Signal transducer and activator of transcription 3), Bcl-3 (B-cell lymphoma 3) and the PI3K (Phosphoinositide 3-kinase)/AKT pathway, whose activities/expression can all be upregulated by CAF-CM. These data suggest that CAF-CM induces Sox2 expression in non-CSCs by activating proteins involved in growth control and drug resistance, leading to higher protection against apoptosis.

Protein­ and growth­modulatory effects of carcinoma­associated fibroblasts on breast cancer cells: Role of interleukin­6.

Carcinoma­associated fibroblasts (CAFs) secrete factors that increase the expression and/or activities of proteins in breast cancer cells and induce resistance to anti­estrogens, such as fulvestrant. A major factor is interleukin­6 (IL­6). This study demonstrated that, across estrogen receptor (ER)α­positive and ­negative cell lines, recombinant human IL­6 (rhIL­6) mimicked most of the CAF­conditioned medium (CM)­induced changes in protein expression patterns; however, in most cases, it failed to recapitulate CAF­CM­triggered alterations in ERK1/2 and AKT activities. The ability of rhIL­6 to induce fulvestrant resistance was dependent upon the culture conditions. In 3D, but not in 2D cultures, rhIL­6 increased the survival of fulvestrant­treated cells, although not to the same extent as observed with CAF­CM. In 2D cultures, rhIL­6 acted in a pro­apoptotic manner and decreased the expression of ATP­binding cassette transporter G2 (ABCG2). The inhibition of the PI3K/AKT pathway had similar effects on apoptosis and ABCG2 expression, linking the failure of rhIL­6 to induce fulvestrant resistance to its inability to activate the PI3K/AKT pathway. In 3D cultures, both CAF­CM and rhIL­6 acted in an anti­apoptotic manner. These activities are likely independent on the PI3K/AKT pathway and ABCG2. Experiments on ERα­negative breast cancer cells revealed a growth­inhibitory effects of both CAF­CM and rhIL­6, which coincided with a reduction in the c­Myc level. These data suggest that IL­6 plays a role in several effects of CAF­CM, including alterations in protein expression patterns, fulvestrant resistance in 3D cultures and growth inhibition. By contrast, IL­6 is unlikely to be responsible for the CAF­CM­induced activation of the PI3K/AKT pathway and fulvestrant resistance in 2D cultures.

Breast cancer stem cells: Features, key drivers and treatment options.

The current view is that breast cancer is a stem cell disease characterized by the existence of cancer cells with stem-like features and tumor-initiating potential. These cells are made responsible for tumor dissemination and metastasis. Common therapies by chemotherapeutic drugs fail to eradicate these cells and rather increase the pool of cancer stem cells in tumors, an effect that may increase the likelyhood of recurrence. Fifteen years after the first evidence for a small stem-like subpopulation playing a major role in breast cancer initiation has been published a large body of knowledge has been accumulated regarding the signaling cascades and proteins involved in maintaining stemness in breast cancer. Differences in the stem cell pool size and in mechanisms regulating stemness in the different breast cancer subtypes have emerged. Overall, this knowledge offers new approaches to intervene with breast cancer stem cell activity. New options are particularly needed for the treatment of triple-negative breast cancer subtype, which is particularly rich in cancer stem cells and is also the subtype for which specific therapies are still not available.

Long-term exposure to carcinoma-associated fibroblasts makes breast cancer cells addictive to integrin ß1.

We studied the long-term effect of stromal factors on the development of fulvestrant-resistance (FR) and fulvestrant-induced dormancy (D). Sublines established from stroma-treated FR-cells (C-FR cells) and D-cells (C-D cells) show permanently high expression of integrin ß1 as well as Bcl-3 and P-STAT3 (C-FR) or IGF1R (C-D). Yet, cells fail to withstand fulvestrant better and do not migrate or grow faster than control cells. Instead, C-D cells rely on stromal factors to perform as well as control cells. In addition, C-FR cells adapted to integrin ß1 for growth in 3D cultures. These data suggest that long-term exposure to stromal factors leads to addiction rather than better performance in cellular activities. We also found that morphologically distinct breast cancer cell line subpopulations share key responses to stromal factors suggesting that intratumoral heterogeneity may play a minor role in the interaction between breast cancer and stromal cells.

Mechanisms governing metastatic dormancy in breast cancer.

Breast cancer is a systemic disease characterized by early dissemination of tumor cells to distant organs. In this foreign environment, tumor cells may stay in a dormant state as single cells or as micrometastases for many years before growing out into a macrometastatic lesion. As metastasis is the primary cause for breast cancer-related death, it is important to understand the mechanisms underlying the maintenance of dormancy and dormancy escape to find druggable targets to eradicate metastatic tumor cells. Metastatic dormancy is regulated by complex interactions between tumor cells and the local microenvironment. In addition, cancer-directed immunity and systemic instigation play a crucial role.

Expression of transmembrane protein 26 (TMEM26) in breast cancer and its association with drug response.

We have previously shown that stromal cells desensitize breast cancer cells to the anti-estrogen fulvestrant and, along with it, downregulate the expression of TMEM26 (transmembrane protein 26). In an effort to study the function and regulation of TMEM26 in breast cancer cells, we found that breast cancer cells express non-glycosylated and N-glycosylated isoforms of the TMEM26 protein and demonstrate that N-glycosylation is important for its retention at the plasma membrane. Fulvestrant induced significant changes in expression and in the N-glycosylation status of TMEM26. In primary breast cancer, TMEM26 protein expression was higher in ERα (estrogen receptor α)/PR (progesterone receptor)-positive cancers. These data suggest that ERα is a major regulator of TMEM26. Significant changes in TMEM26 expression and N-glycosylation were also found, when MCF-7 and T47D cells acquired fulvestrant resistance. Furthermore, patients who received aromatase inhibitor treatment tend to have a higher risk of recurrence when tumoral TMEM26 protein expression is low. In addition, TMEM26 negatively regulates the expression of integrin ß1, an important factor involved in endocrine resistance. Data obtained by spheroid formation assays confirmed that TMEM26 and integrin ß1 can have opposite effects in breast cancer cells. These data are consistent with the hypothesis that, in ERα-positive breast cancer, TMEM26 may function as a tumor suppressor by impeding the acquisition of endocrine resistance. In contrast, in ERα-negative breast cancer, particularly triple-negative cancer, high TMEM26 expression was found to be associated with a higher risk of recurrence. This implies that TMEM26 has different functions in ERα-positive and -negative breast cancer.

Stromal cells promote anti-estrogen resistance of breast cancer cells through an insulin-like growth factor binding protein 5 (IGFBP5)/B-cell leukemia/lymphoma 3 (Bcl-3) axis.

There is strong evidence that stromal cells promote drug resistance of cancer. Here, we show that mesenchymal stem cells (MSCs) and carcinoma-associated fibroblasts (CAFs) desensitize ERα-positive breast cancer cells to the anti-estrogen fulvestrant. In search for the mechanism, we found that MSCs and CAFs similarly increased the activity of the PI3K/AKT and the JAK/STAT3 pathways and upregulated the expression of integrin ß1, IGF1R, HIF1α, CAIX and Bcl-3 in MCF-7 cells. Further analyses revealed that MSCs and CAFs coordinately induce these changes by triggering the downregulation of IGFBP5. Loss of IGFBP5 in MCF-7 cells was an early and long-lasting event in response to MSCs and CAFs and was accompanied by growth stimulation both in the absence and presence of fulvestrant. The growth-stimulatory effect in the absence of fulvestrant could be attributed to PI3K/AKT pathway activation and could be mimicked by insulin. The growth-promoting effect in the presence of fulvestrant depended upon the upregulation of Bcl-3. By cRNA microarray analysis we identified additional IGFBP5 targets, of which two (KLHL4 and SEPP1) were inversely regulated by IGFBP5 and Bcl-3. BT474 cells also responded to stromal cells by downregulating IGFBP5 and upregulating the P-AKT, Bcl-3 and IGF1R levels, whereas T47D cells did not show any of these responses. In conclusion, our data suggest that, by targeting IGFBP5 expression in ERα-positive breast cancer cells, such as MCF-7 cells, MSCs and CAFs are able to orchestrate a variety of events, particularly activation of the PI3K/AKT pathway, upregulation of Bcl-3 expression and desensitization to anti-estrogen.

The role of the transcription factor Ets1 in carcinoma.

Ets1 belongs to the large family of the ETS domain family of transcription factors and is involved in cancer progression. In most carcinomas, Ets1 expression is linked to poor survival. In breast cancer, Ets1 is primarily expressed in the triple-negative subtype, which is associated with unfavorable prognosis. Ets1 contributes to the acquisition of cancer cell invasiveness, to EMT (epithelial-to-mesenchymal transition), to the development of drug resistance and neo-angiogenesis. The aim of this review is to summarize the current knowledge on the functions of Ets1 in carcinoma progression and on the mechanisms that regulate Ets1 activity in cancer.

The impact of tumor stroma on drug response in breast cancer.

In the last two decades the breast cancer mortality rate has steadily declined, in part, due to the availability of better treatment options. However, drug resistance still remains a major challenge. Resistance can be an inherent feature of breast cancer cells, but can also arise from the tumor microenvironment. This review aims to focus on the modulatory effect of the tumor microenvironment on the differing response of breast cancer subtypes to targeted drugs and chemotherapy.

Paracrine effects of stem cells in wound healing and cancer progression (Review).

Stem cells play an important role in tissue repair and cancer development. The capacity to self-renew and to differentiate to specialized cells allows tissue-specific stem cells to rebuild damaged tissue and cancer stem cells to initiate and promote cancer. Mesenchymal stem cells, attracted to wounds and cancer, facilitate wound healing and support cancer progression primarily by secreting bioactive factors. There is now growing evidence that, like mesenchymal stem cells, also tissue-specific and cancer stem cells manipulate their environment by paracrine actions. Soluble factors and microvesicles released by these stem cells have been shown to protect recipient cells from apoptosis and to stimulate neovascularization. These paracrine mechanisms may allow stem cells to orchestrate wound healing and cancer progression. Hence, understanding these stem cell-driven paracrine effects may help to improve tissue regeneration and cancer treatment.

Cancer stem cells in breast cancer.

There is increasing evidence that cancer stem cells (CSCs) play a critical role in breast cancer initiation, progression, metastasis and drug resistance. It is thought that they are either generated from normal mammary stem/progenitor cells or from mammary epithelial cells by epithelial-mesenchymal transition. Breast CSCs are characterized by the activation of stemness-related pathways, such as the Notch and Wnt pathways, and by the expression of certain stem cell markers, such as CD44, EpCAM and ALDH1. CSCs form a minor population, whose proportion depends on various factors, including environmental conditions. Since CSCs are highly resistant to chemotherapy, additional treatment of breast cancer patients with CSC-specific drugs, such as salinomycin and gamma-secretase inhibitors which target the Wnt or Notch pathway, respectively, will be required. Interestingly, an equilibrium seems to exist between CSCs and non-stem cancer cells, and there are indications that CSCs can be recruited from non-stem cancer cells. As a consequence, it may be necessary to combine a therapy targeting CSCs with common chemotherapy that targets the bulk tumor to avoid the regeneration of CSCs.

Cyclic AMP enhances TGFß responses of breast cancer cells by upregulating TGFß receptor I expression.

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFß and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFß-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TßRI (TGFß receptor 1), were responsive to cAMP. While YAP had little effect on TGFß-dependent expression and Smad3 phosphorylation, a constitutively active form of TßRI mimicked the cAMP effect on TGFß signaling. In 3D-cultured cells, which show much higher levels of TßRI and cAMP, TßRI was unresponsive to cAMP. Upregulation of TßRI expression by cAMP was dependent on transcription. A proximal TßRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TßRI expression at least partially by activating TßRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TßRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TßRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFß on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFß pathways. In summary, these data suggest that combined effects of cAMP and TGFß, as e.g. induced by mesenchymal stem cells, involve the upregulation of TßRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

Inverse expression of cystein-rich 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) in borderline tumors and carcinomas of the ovary.

Members of the CCN [cystein-rich 61 (Cyr61)/connective tissue growth factor (CTGF)/nephroblastoma (NOV)] protein family are involved in the regulation of cellular proliferation, apoptosis, and migration and are also assumed to play a role in carcinogenesis. Therefore, we performed a retrospective study to investigate the immunohistochemical expression of both Cyr61 and CTGF in 92 borderline tumors (BOTs) and 107 invasive carcinomas of the ovary (IOCs). To determine their diagnostic and prognostic value, we correlated protein expression with clinicopathologic factors including overall and disease-free survival. Cyr61 and CTGF were found to be inversely expressed in both BOTs and IOCs, with a stronger expression of Cyr61 in IOCs. Moreover, Cyr61 was found to be preferentially expressed in high-grade serous carcinomas, whereas CTGF was found more frequently in low-grade serous carcinomas. Weak Cyr61 levels correlated with both low estrogen receptor and p53 expression (P=0.038, P=0.04, respectively). However, no association was observed between CTGF, estrogen receptor, and p53 expression levels in IOCs. Regarding prognosis, Cyr61 was found to be of no value, but the loss of CTGF was found to be associated with a poor prognosis in multivariate analysis of overall (relative risk 2.8; P=0.050) and disease-free (relative risk 2.3; P=0.031) survival. Cyr61 and CTGF are inversely expressed in BOTs and IOCs, and loss of CTGF independently indicates poor prognosis in IOCs.

Highly sensitive profiling of CD44+/CD24- breast cancer stem cells by combining global mRNA amplification and next generation sequencing: evidence for a hyperactive PI3K pathway.

We performed next generation sequencing- and microarray-based gene expression profiling of CD44(+)/CD24(-)/CD45(-) breast CSCs (cancer stem cells) isolated from primary ERα-positive breast cancer. By combining semi-automated dissociation of human tumor tissue, magnetic cell sorting and cDNA amplification less than 500 CSCs were required for transcriptome analyses. Besides overexpressing genes involved in maintenance of stemness, the CSCs showed higher levels of genes that drive the PI3K pathway, including EGFR, HB-EGF, PDGFRA/B, PDGF, MET, PIK3CA, PIK3R1 and PIK3R2. This suggests that, in CSCs of ERα-positive breast cancer, the PI3K pathway which is involved in endocrine resistance is hyperactive.

Protein expression of the Ets transcription factor Elf-1 in breast cancer cells is negatively correlated with histological grading, but not with clinical outcome.

Several members of the Ets (E26 transformation specific) transcription factor family are involved in tumor progression, e.g. by activating matrix metalloproteases. Ets proteins share a unique DNA-binding domain, the Ets domain, which specifically recognizes GGAA/T-containing sequences common in many promoters. While the roles of quite a number of Ets proteins in carcinogenesis have been well established, little is known about the importance of the Ets protein Elf-1 (E74-like factor 1) in cancer. Herein, we analyzed the expression of Elf-1 in breast cancer. We found that, like T-cells, breast cancer cells express both the 80 and 98 kDa isoforms of the Elf-1 protein with the 98 kDa isoform only be present in the nucleus. Immunohistochemical analysis of 119 breast cancer biopsies showed anti-Elf-1 immunoreactivity exclusively in the nucleus. Elf-1 expression varied largely among the breast cancer samples showing a negative correlation with histological grading. However, no association of Elf-1 expression with clinical outcome was observed, even when sub-cohorts of patients who received either only adjuvant endocrine treatment or only chemotherapy were separately analyzed. These data suggest that Elf-1 may modulate breast cancer progression to some extent without having an impact on survival of breast cancer patients.

Mesenchymal stem cells and carcinoma-associated fibroblasts sensitize breast cancer cells in 3D cultures to kinase inhibitors.

Stromal cells, such as mesenchymal stem cells (MSCs) and carcinoma-associated fibroblasts (CAFs), play a role in cancer progression. To analyze their ability to modulate drug response, we generated spheroids of MCF-7 or MDA-MB-231 breast cancer cells in the absence or presence of human (h)MSCs or hCAFs and tested the susceptibility of the breast cancer cells to three different kinase inhibitors (TKI258, RAD001 and RAF265) used in cancer therapy. While stromal cells did not affect the response of either breast cancer cell line to the PDGFR/FGFR/VEGFR inhibitor TKI258, they sensitized breast cancer cells to the mTOR inhibitor RAD001. In MCF-7 cells, this was accompanied by increased apoptosis. hMSCs and to a lesser extent hCAFs also enhanced the cytotoxic effect of RAF inhibitor RAF265 on MDA-MB-231 cells. Searching for the mechanism that underlies the effect of stromal cells on RAF265 response we found that stromal cells inhibited RAF265-induced increase in ERK1/2 phosphorylation, supported RAF265-dependent downregulation of PKCα (protein kinase Cα) and prevented RAF265-induced conversion of LC3B, a marker of autophagy. To mimic the changes in ERK1/2 phosphorylation and PKCα expression in response to the stromal cells, we treated cells with MEK1 inhibitor U0126 or PKCα inhibitor Gö6976, respectively. U0126, but not Gö6976, was as effective as hMSCs in sensitizing MDA-MB-231 cells to RAF265. This suggests that hMSCs and hCAFs increased the cytotoxic effect of RAF265 on MDA-MB-231 cells by downregulating ERK1/2 phosphorylation. In summary, this study shows that hMSCs are able to render breast cancer cells more susceptible to kinase inhibitors and that, to the most part, hCAFs to which hMSCs can differentiate are able to mimic the drug-sensitizing effects of hMSCs.