Colorectal cancer is one of the most common causes of cancer-related mortality worldwide [1
]. Patients are conventionally treated with surgery, chemotherapy and radiotherapy. However, about 50% of patients are diagnosed at late stage and develop liver metastasis, which leads to the low survival rate [3
]. In patients with metastatic colorectal cancer, the combination treatment of 5-fluorouracil (5-FU), Folinic acid and Oxaliplatin (FOLFOX) (5-FU), Folinic acid and Irinotecan (FOLFIRI) is employed and improves overall survival in some patents. However, most patients develop a drug resistance during the course of treatment [4
]. Therefore, understanding the mechanisms underlying the resistance is essential for the development of effective treatments.
Recent studies have gradually revealed that cancer stem cells are associated with chemoresistance of colorectal cancer [5
]. Cancer stem cells exhibit alterations of DNA repair and express ABC membrane transporters mediating chemoresistance [6
]. Since many tumors including colorectal cancer might progress due to the cancer stem cells that are not sensitive to the treatment, there is an emerging need for novel therapies targeting cancer stem cells.
Cancer stem cells use various signal pathways such as Wnt, Notch and Hedgehog [6
]. Recently, inhibition of Notch signals has been identified as an approach to colorectal cancer [7
]. Small molecule antagonists targeting Wnt signals also inhibited proliferation of colon cancer cells [8
]. Furthermore, combination treatment with Notch signal inhibitor and Irinotecan reduced tumor growth [9
]. These reports imply that inhibition of cancer stem cell-related signals could be useful for the effective treatments through upregulating the sensitivity for anti-cancer drugs. Nevertheless, the detailed mechanisms remain unclear owing to the lack of proper culture model for cancer stem cells from colorectal cancer patients.
In the previous study, we established three-dimensional (3D) organoid model from tumor colorectal tissues of human patients using an air–liquid interface (ALI) method [10
]. Tumor ALI organoids consisted of both epithelial and mesenchymal components and closely recapitulated epithelium structures of the original tumor. In addition, they contained numerous cancer stem cells expressing LGR5 and CD44. These characteristics indicate that they could recapitulate tumor microenvironment in the 3D culture. Using this model, we for the first time demonstrated that tumor ALI organoids are more resistant to toxicity of 5-FU and Irinotecan than colorectal cancer cell lines, such as SW480, SW620 and HCT116 [10
Nevertheless, the resistant mechanisms remain unclear. Here, we investigated whether inhibitors for cancer stem cell-related signal improve the anti-cancer drug resistance of organoids. Among several signal inhibitors, the resistance of tumor ALI organoids is improved by the combination treatment with Hedgehog signal inhibitors and anti-cancer drugs through the decrease of their stemness. The combination treatment also prevents colony formation of colorectal cancer cell lines.
The major findings of the present study are as follows: (1) Hedgehog signal inhibitors were more effective on decreasing the cell viability of tumor organoids compared with Wnt and Notch signal inhibitors (Figure 1
). (2) The combination treatment with Hedgehog signal inhibitor and 5-FU, Irinotecan or Oxaliplatin effectively decreased the cell viability of tumor organoids (Figure 2
). (3) In tumor organoids, expression of c-Myc, CD44 and Nanog was inhibited by Hedgehog signal inhibitors likely through the decrease of GLI-1 expression (Figure 3
). (4) The combination treatment with Hedgehog inhibitor and 5-FU or Irinotecan prevented colony formation of HCT116 and SW480 (Figure 4
). Collectively, our results indicate that Hedgehog signals regulate anti-cancer drug resistance of human colorectal tumor patient-derived ALI organoids (Figure 5
) and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer.
Resistance to anti-cancer drugs in colorectal cancer patients is a major problem. GLI-1 is known to be vital in cancer biology and overexpressed in colorectal cancer cells [14
]. Recent study showed that GLI-1 expression was elevated in 5-FU resistant colorectal cancer cell line LoVo-R compared with non-resistant one [17
]. In the same report, it was also shown that knockdown of GLI-1 gene decreased the resistance to 5-FU. Nevertheless, there was no evidence showing that GLI-1 mediates anti-cancer drug resistance in primary colorectal cancer cells. In the present study, we for the first time demonstrated that Hedgehog signal inhibitor decreased the resistance to 5-FU, Irinotecan and Oxaliplatin likely through the inhibition of GLI-1 expression using colorectal cancer patient-derived ALI organoids (Figure 2
and Figure 3
). Although the detailed mechanisms by which GLI-1 regulates the resistance remain unclear, further studies by using the ALI organoid system might contribute to overcoming chemoresistance in colorectal cancer patients.
Tumor microenvironment mediates cancer initiation, progression and metastasis [18
]. In pancreatic cancer, Hedgehog signals are activated in the stromal cells rather than epithelial cells, which supports tumorigenesis [19
]. In pancreatic cancer model mice, treatment with a Hedgehog signal inhibitor, IPI-926 prevented the tumor progression through the depletion of stromal cells [21
]. In colorectal cancer model mice, epithelial cells secrete Hedgehog ligands to maintain the stromal phenotype, which is required for adenoma development [22
]. These reports suggest that Hedgehog ligands secreted from tumor cells stimulate the stromal cells to secrete tumor growth factors. In the present study, we showed that Hedgehog signals mediate the chemoresistance of tumor ALI organoids (Figure 2
). Since our ALI tumor organoid model contains both epithelial and stromal components, it might become a useful tool to clarify the interacting mechanisms between epithelial and mesenchymal cells, which regulate the resistance of anti-cancer drugs in colorectal cancer.
Cancer stem cells possess the potential of self-renewal, multi-lineage differentiation and tumorigenicity. In the colorectal cancer treatment, cancer stem cells are known to be associated with resistance to chemotherapy and radiotherapy, which causes the recurrence of cancer and promotes metastasis [23
]. Colorectal cancer stem cells express several cell surface markers, such as CD44, CD24, CD133 and CD146 [26
]. The relationship between cancer stem cell markers and Hedgehog signaling has been gradually clarified. For examples, expression of cancer stem cell markers correlated with activation of Hedgehog signals in gemcitabine-resistant pancreatic cancer cells [27
]. It was also reported that expression level of Hedgehog-related proteins was higher in gastric cancer stem cells expressing both CD44 and CD24 compared with non-expressing ones [28
]. In the present study, we showed that Hedgehog inhibitor increased the sensitivity for anti-cancer drugs (Figure 2
) and decreased expression of CD44, c-Myc and Nanog likely through the inhibition of GLI-1 in tumor ALI organoids (Figure 3
). These results indicate that combination therapy of Hedgehog inhibitor with anti-cancer drugs might become a promising strategy to remove colorectal cancer stem cells from the patients.
Survival rate in metastatic colorectal cancer patients has been improved due to major advances in chemotherapy and targeted drugs. Since several studies showed that dysregulation of Hedgehog signals mediates colorectal cancer progression and metastasis [29
], Hedgehog signals are regarded as a new therapeutic target for the treatment of colorectal cancer. Hedgehog signal-related protein, SMO regulates nuclear translocation of GLI-1 that promotes transcription of target genes, such as c-Myc and CD44 [11
]. Inhibition of SMO has been studied in a variety of tumor types [30
]. However, a SMO inhibitor, Vismodegib, did not extend progression-free survival in colorectal cancer patients [31
]. In addition, acquired resistance to SMO inhibitors also occurred in the clinical test phase [33
]. To overcome these problems, more specific and effective inhibitor for Hedgehog signals is required. GANT61 is a small molecule that inhibits binding of GLI-1 and induces DNA double strand breaks [34
]. In the present study, we showed that a SMO inhibitor, AY9944 and a GLI-1 inhibitor, GANT61 improved the sensitivity for anti-cancer drugs in tumor ALI organoids (Figure 3
). We also showed that combination of AY9944 or GANT61 with 5-FU or Irinotecan prevented the colony formation in SW480 and HCT116 cells (Figure 4
). These results indicate that combination of not only SMO inhibitor but also selective GLI-1 inhibitor with anti-cancer drugs might be useful for an effective combinational therapy of colorectal cancer.
In summary, we for the first time demonstrated that Hedgehog signals mediate the resistance to anti-cancer drugs in human colorectal tissue-derived ALI organoids through the decrease of their stemness. It was also suggested that combinational therapy of Hedgehog inhibitor with anti-cancer drugs is effective for colony formation of colorectal cancer cell lines. Further studies on the relationship between Hedgehog signal and chemotherapy contribute to developing new strategy for colorectal cancer treatment.