No abstract

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 β1, 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.

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.

Carcinoma-associated fibroblasts (CAFs) secrete factors that increase the expression and/or activities of proteins in breast cancer cells and induce resistance to anti-estrogens, such as fulvestrant. A major factor is interleukin-6 (IL-6). This study demonstrated that, across estrogen receptor (ER) α-positive and -negative cell lines, recombinant human IL-6 (rhIL-6) mimicked most of the CAF-conditioned medium (CM)-induced changes in protein expression patterns; however, in most cases, it failed to recapitulate CAF-CM-triggered alterations in ERK1/2 and AKT activities. The ability of rhIL-6 to induce fulvestrant resistance was dependent upon the culture conditions. In 3D, but not in 2D cultures, rhIL-6 increased the survival of fulvestrant-treated cells, although not to the same extent as observed with CAF-CM. In 2D cultures, rhIL-6 acted in a pro-apoptotic manner and decreased the expression of ATP-binding cassette transporter G2 (ABCG2). The inhibition of the PI3K/AKT pathway had similar effects on apoptosis and ABCG2 expression, linking the failure of rhIL-6 to induce fulvestrant resistance to its inability to activate the PI3K/AKT pathway. In 3D cultures, both CAF-CM and rhIL-6 acted in an anti-apoptotic manner. These activities are likely independent on the PI3K/AKT pathway and ABCG2. Experiments on ERα-negative breast cancer cells revealed a growth-inhibitory effects of both CAF-CM and rhIL-6, which coincided with a reduction in the c-Myc level. These data suggest that IL-6 plays a role in several effects of CAF-CM, including alterations in protein expression patterns, fulvestrant resistance in 3D cultures and growth inhibition. By contrast, IL-6 is unlikely to be responsible for the CAF-CM-induced activation of the PI3K/AKT pathway and fulvestrant resistance in 2D cultures.

Abstract. 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.

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ 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 TGFβ-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 TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI 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 TGFβ 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 TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

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 β1, an important factor involved in endocrine resistance. Data obtained by spheroid formation assays confirmed that TMEM26 and integrin β1 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.

Background: Bone-derived mesenchymal stem cells (MSCs) are attracted to cancer lesions and may differentiate to CAFs. By interacting with cancer cells, MSCs and CAFs may promote cancer progression and modulate drug sensitivity. Material and Methods: To analyze ability of MSCs and CAFs 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) as used in cancer therapy. Results: While MSCs and CAFs did not affect the response of either breast cancer cell line to 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 PKCalpha (protein kinase Calpha) and prevented RAF265-induced conversion of LC3B, a marker of autophagy. To mimic the changes in ERK1/2 phosphorylation and PKCalpha expression in response to the stromal cells, we treated cells with MEK1 inhibitor U0126 or PKCalpha inhibitor Gö6976, respectively. U0126, but not Gö6976, was as effective as hMSCs in sensitizing MDA-MB-231 cells to RAF265. Discussion: Our data suggest 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. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-08.