The role of tumor-proximal factors in tumor plasticity during chemoresistance and metastasis following chemotherapy is well studied. However, the role of endothelial cell (EC) derived paracrine factors in tumor plasticity, their effect on chemotherapeutic outcome, and the mechanism by which these paracrine factors modulate the tumor microenvironment are not well understood. In this study, we report a novel mechanism by which endothelial miR-125a and let-7e-mediated regulation of interleukin-6 (IL-6) signaling can manipulate vasculogenic mimicry (VM) formation of MDA-MB-231 breast cancer cells. We found that endothelial IL-6 levels were significantly higher in response to cisplatin treatment, whereas levels of IL-6 upon cisplatin exposure remained unchanged in MDA-MB-231 breast cancer cells. We additionally found an inverse correlation between IL-6 and miR-125a/let-7e expression levels in cisplatin treated ECs. Interestingly,
Although many chemotherapeutic drugs have received remarkable initial responses, tumor recurrence is common. Chemoresistance and metastasis are the most common causes of cancer related death (1–3). In light of the limitations of current chemotherapeutic drugs, identification of novel mechanisms to overcome tumor recurrence, chemoresistance, and metastasis is critical.
To date, most studies have focused on the role of tumor-proximal factors in tumor recurrence and chemoresistance following chemotherapy (1, 4–6). Despite the support provided by the surrounding tumor microenvironment to cancer cells, the role of tumor stromal cells such as endothelial cells (ECs) in chemotherapeutic outcomes is still poorly understood. Healthy vascular ECs play an essential role in the maintenance of vascular homeostasis in response to various stimuli (7–9). In contrast, endothelial dysfunction caused by chemotherapeutic drugs contributes to tumor growth and survival (10, 11). However, it is not well understood how EC-derived paracrine factors affect endothelial dysfunction following chemotherapy, and how they influence tumor plasticity which eventually causes chemoresistance. Vasculogenic mimicry (VM) refers to the formation of tumor cell-lined endothelium-like vessels, which is known to provide blood supply for tumor growth and represents a key link connecting the aggressiveness of cancer and metastasis with short survival of patients (12–15). In addition, emerging evidence shows that VM plays key roles in tumor plasticity during drug resistance following chemotherapy (16, 17).
Interleukin-6 (IL-6) signaling plays key roles in the development of chemoresistance and in recurrence of various cancers. (18–20). Thus,
In this study, we sought to define the role of the EC-derived paracrine factor, IL-6 in tumor plasticity in a chemotherapeutic therapy, and to understand the role of miRNAs in IL-6 signaling mediated paracrine effects on tumor cells. We demonstrated that IL-6 levels are upregulated as a consequence of decrease in miR-125a and let-7e expression in cisplatin treated ECs, although IL-6 levels were not affected in cisplatin treated MDA-MB-231 breast cancer cells, which suggests the importance of endothelium-derived IL-6. We found that conditioned medium (CM) from cisplatin treated ECs induces significantly greater formation of VM in MDA-MB-231 breast cancer cells, and might eventually cause drug resistance and extensive metastasis. These findings reveal a novel mechanism by which miRNA-mediated regulation of endothelial IL-6 signaling can manipulate VM formation through the platinum-containing chemotherapeutic agent, cisplatin in breast cancer cells.
To analyze the effect of a chemotherapeutic drug on IL-6 expression in the tumor microenvironment, we treated tumor cells and ECs with cisplatin and assessed expression levels of IL-6. Treatment of human umbilical vein ECs (HUVECs) with cisplatin led to significantly increased IL-6 expression levels (Fig. 1A), whereas IL-6 expression was not affected in cisplatin treated MDA-MB-231 breast cancer cells (Data not shown), suggesting a potential role for endothelium-derived paracrine IL-6 secretion in the microenvironment of breast cancer. Several molecular mechanisms have been shown to affect IL-6 expression, including a critical role for miRNA-mediated regulation in different cellular contexts, such as in macrophages (22) and preosteoblasts (23). The potential role of miRNAs as regulators of IL-6 expression in ECs is still unknown. Thus, to identify miRNAs that might regulate the expression of IL-6 in response to cisplatin in ECs, we first analyzed the expression of miRNAs in response to cisplatin in HUVEC and EC-enriched miRNAs, using publicly available miRNA expression profiling data (22, 23). The target prediction algorithm (TargetScan) was used to identify potential miRNAs targeting IL-6 and related genes (Fig. 1B). From these results, we found two miRNAs (miR-125a and let-7e) which are highly expressed in ECs and are expressed as a cluster. We next examined miR-125a and let-7e expression in cisplatin treated ECs. As shown in Fig. 1C, miR-125a and let-7e levels decreased in response to cisplatin treatment in ECs. These findings suggest that miR-125a and let-7e may be involved in the mechanism underlying the induction of IL-6 expression in response to cisplatin.
To further investigate the relationship between miR-125a/let-7e and IL-6, we determined the effects of miR-125a and let-7e overexpression on IL-6 expression in HUVECs. Overexpression of miR-125a or let-7e led to significant downregulation of IL-6 expression, whereas inhibition of endogenous miR-125a or let-7e by anti-miRs in HUVECs led to upregulation of IL-6 expression (Fig. 2A). Next, we determined whether miR-125a and let-7e regulate IL-6 expression by binding directly to its 3′UTR. Let-7e was predicted to target the
We further examined additional predicted targets of let-7e and miR-125a which might play an essential role in the IL-6 signaling pathway in ECs. As shown in Fig. 2C and 2D, overexpression or knockdown of miR-125a or let-7e had effects on IL-6R and STAT3 expression similar to those on IL-6 expression, although let-7e had no effect on
Because the IL-6 signaling pathway plays a critical role in vascular functions such as inflammation and monocyte adhesion (25, 26), we examined the effect of miR-125a/let-7e on IL-6 induced adhesion of monocytes to ECs. miR-125 and let-7e overexpression significantly inhibited the IL-6 induced THP-1 cell adhesion to HUVECs (Fig. 3A), suggesting that this effect may be secondary to the targeting of IL-6R and STAT3. However, miR-125 and let-7e overexpression had no effect on the TNF-α induced THP-1 cell adhesion to HUVECs. These results indicate that miR-125 and let-7e might act specifically on the adhesion of monocytes to ECs in response to IL-6.
Next, to understand the role of EC derived paracrine IL-6 on tumor plasticity, we treated MDA-MB-231 breast cancer cells with IL-6, and examined the formation of VM using the well established
Previous studies have associated elevated IL-6 levels with poor overall survival and tumor progression in various cancers (27–29). In addition, IL-6 signaling plays a key role in the development of chemoresistance in various cancers (18–20). Vasculature is essential for cancer progression and reprograms the tumor microenvironment. In particular, ECs play a critical role in the maintenance of vascular homeostasis, whereas dysfunctional ECs contribute to the pathogenesis of various diseases and to tumor progression by secreting cytokines. (10, 11). Here, we describe miRNA-driven regulation of IL-6 signaling in ECs and show its involvement in the regulation of cell adhesion and in the formation of VM in an autocrine and paracrine manner in tumor microenvironments in response to cisplatin treatment.
Many studies have shown that miRNAs play key roles in the context of cancer and vascular biology (9, 10, 30–33). However, endothelial miRNA-mediated autocrine and paracrine regulation of chemotherapeutic outcome in tumor microenvironments is poorly understood. Previous reports show that miR-125a and let-7e are significantly downregulated in many cancers, and downregulation of these miRNAs leads to enhanced tumor progression, chemoresistance, and metastasis despite strong context dependence of the role of miR-125a and let-7e in cancer (34–40). Although miR-125a and let-7e are highly expressed in ECs (8, 41), and have been studied in different cellular contexts such as in immune cells, preosteoblasts, and in a variety of cancers (23, 24, 34, 37, 42, 43), their role in EC biology remains unclear. Therefore, it is important to understand the role or regulation of miR-125a and let-7e in tumors as well as in tumor microenvironments such as ECs. Our current findings provide three key aspects of highly enriched endothelial miRNAs, miR-125a and let-7e, which might act as important mediators for modulating tumor plasticity and thus affect the chemotherapeutic outcome. First, we found that IL-6 and miR-125a/let-7e are inversely regulated by the platinum-containing chemotherapeutic agent, cisplatin in HUVECs. Second, we showed that miR-125a and let-7e directly target
VM reflects tumor plasticity of aggressive tumor cells acquiring tumor cell-lined endothelium-like vessels, and is an independent blood supply system from endothelial vessels. Emerging evidence shows that the roles of VM in several cancers, are associated with tumor aggressiveness and poor patient survival, as well as tumor plasticity during chemoresistance and metastasis (12–17). In light of the association of VM and tumor progression, coupled with ineffective anti-angiogenic drugs associated with the inhibition of VM (44, 45), identifying novel therapeutic targets that inhibit VM, might be effective to overcome chemoresistance and to eliminate tumor progression. However, the molecular mechanism underlying the formation of VM in tumor microenvironments remains unclear. Our current data provide a novel mechanism by which restoration of miR-125a and let-7e expression in cisplatin treated ECs can inhibit VM formation in MDA-MB-231 breast cancer cells, in a paracrine manner via targeting IL-6 signaling in ECs. These findings support the emerging role of dysfunctional EC-tumor cell crosstalk which likely plays a critical role in tumor progression and chemoresistance. Given these findings, future studies will be necessary to identify other paracrine factors in ECs affecting chemotherapeutic outcome. Furthermore, our ongoing studies to elucidate the precise role of miR-125/let-7e-IL-6 signaling axis in ECs, both in chemotherapeutic drug induced endothelial dysfunction, and tumor progression in a paracrine manner, will provide greater insights into tumor-EC interaction.
In conclusion, we show that downregulation of miR-125a and let-7e and the subsequent activation of IL-6 signaling in ECs on chemotherapeutic drugs is closely associated with cisplatin-induced VM formation and monocyte adhesion in tumor microenvironments. These findings might promote the development of novel therapeutic candidate drugs, such that chemotherapy combined with IL-6 inhibition might help to overcome chemoresistance and, thus, help in treatment of cancer.
An extended Materials and Methods section is available in the
HUVECs (Human umbilical vein ECs; Lonza and Yale VBT Core) were maintained in EBM-2 basal medium supplemented with EGM-2 (Lonza) with 1% penicillin/streptomycin (Welgene). HUVECs were grown to 70% to 90% confluency and used between passages 4 to 7 for all experiments. MDA-MB-231 (human breast adenocarcinoma cells) were maintained in DMEM (Dulbecco’s modified Eagles medium; Welgene) supplemented with 10% FBS (fetal bovine serum, Hyclone) and 1% penicillin-streptomycin (Welgene). HEK-293 cells (human embryo kidney cells) were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained in a 5% CO2 incubator at 37°C. siRNA (Bioneer), miRNA mimics and anti-miRs (miRVana; Ambion) were transfected into cells using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.
This work was supported by the Medical Research Center Program (No. 2011-0030074) through National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP).