Sulforaphane Inhibits Self-renewal of Lung Cancer Stem Cells Through the Modulation of Polyhomeotic Homolog 3 and Sonic Hedgehog Signaling Pathways

Background: Sulforaphane (SFN), an active compound in cruciferous vegetables has been characterized for its antiproliferative capacity. We investigated the role and molecular mechanism through which SFN regulates proliferation and self-renewal of lung cancer stem cells. Methods: Lung cancer stem cells (CD133-positive cells) were isolated by MACs and then measured by flow cytometry. The ability of cell proliferation was assessed by MTT assays and tumorsphere formation assays. The expressions of Sonic Hedgehog (Shh), Smoothened (Smo), Gli1 and Human Polyhomeotic Homolog 3 (PHC3) in cells were measured by quantitative reverse transcription polymerase chain reaction (qPCR) and western blot assays. The expression of transcription factor SOX2 in lung cancer stem cells was also determined by western blot assay. Shh was knocked down by siRNA to further study the role of SFN and Shh signaling pathways in lung cancer. Results: SFN inhibited the proliferation of lung cancer cells and lung cancer stem cells simultaneously. Meanwhile, we observed that Sonic Hedgehog (SHH) signaling pathway, SOX2 and Polyhomeotic Homolog 3 (PHC3) were highly activated in lung cancer stem cells. Knockdown of Shh led to reduced H460 and A549 cells proliferation. Furthermore, we observed that SFN inhibited the activity of PHC3 and SHH signaling pathways in the lung cancer stem cells. In addition, SFN combined with Knock-down of Shh gene showed a greater effect on the proliferation of lung cancer cells. Conclusion: SFN is an effective new drug which can inhibit proliferation of lung cancer stem cells through the modulation of PHC3 and SHH signaling pathways. It provides a novel target for improving therapeutic efficacy for lung cancer stem cells.

Background Lung cancer is one of the most prevalent cancers which accounts for approximately one-fourth of the cancer incidence and is the second leading cause of death both in China and developed countries around the world [1,2]. Despite initial treatment with conventional therapy has a high remission rate eventually the disease almost always relapses in the form of resistance to chemotherapy and radiation therapy, therefore the overall 5-year survival rate of patients is less than 5% [3].
Increasing evidence of the existence of cancer stem cells(CSCs) in lung cancer explains why standard chemotherapy or radiotherapy regimens against lung cancer are usually ineffective and result in further tumor recurrence and spread [4].CD133 is a cell membrane glycoprotein and it contains five transmembrane rings. It has been reported that CD133-positive subpopulation of multipotent cells has the biological features of cancer stem cells which possess extremely proliferative and self-renewal characteristics [5,6]. Although CSCs are only a small part of tumors, they have a powerful ability to self-renew and indefinite proliferation. The tumors rich in CSCs are more aggressive and lead to worse clinical outcomes. Therefore, the development of therapeutic strategies and drugs that specifically target CSCs can eradicate tumors effectively and reduce the risk of recurrence and metastasis. Sulforaphane (SFN) is an isothiocyanate (ITC) found in cruciferous vegetables such as broccoli and cabbage [7,8]. A series of clinical trials have demonstrated that SFN can inhibit the malignant growth of cancer cells such as pancreatic cancer cells and breast cancer cells and has no obvious toxicity to the normal cells [9][10][11][12]. Therefore, it has been suggested safe to use SFN as a potential candidate for cancer treatment. However, the effect of SFN on the development of lung cancer and its mechanism is still unknown.
Sonic Hedgehog (SHH) signaling pathway has an essential role in the control of stem cell growth in embryonic tissues, and it plays a key role in the development of tissues and organs [13]. 4 Recent evidence suggests that the SHH signaling pathway contributes to tumorigenesis when it is mutated or misregulated [14,15]. Since the SHH pathway plays a critical role in the renewal of cancer stem cells, blockade of SHH has evolved as a promising therapy for various types of cancers including lung cancer [16][17][18]. Sonic hedgehog (Shh), Smoothened (Smo) and Gli1 are important factors in the SHH signaling pathway. Human Polyhomeotic Homolog 3 (PHC3) is a member of the polycomb Group(PcG) protein family [19], and PcG is a family of chromatin-related gene silencing proteins that regulates gene expression program in epigenetics. PcG proteins have essential roles in early embryonic development and have been implicated in embryonic stem cell pluripotency [20]. The PRCs silence tumor-suppressor genes by histone modifications, leading to cancer cell proliferation, metastasis and drug resistance [21]. Thus, PHC3 and SHH signaling pathways have essential roles in controlling stem cell growth in early embryonic development. However, the role of PHC3 and SHH signaling pathways in human lung cancer stem cells still needs to be elucidated.
In this study, we examined the effect of SFN on the proliferation and self-renewal of lung cancer stem cells. Moreover, we analyzed the role of PCH3 and SHH signaling pathways in lung cancer cells and lung cancer stem cells. Furthermore, we analyzed SFN whether it was associated with the proliferation and self-renewal of lung cancer stem cells through the modulation of the PHC3 and SHH signaling pathways.

Cell culture
Human non-small cell carcinoma of the lung cancer A549 and H460 cell lines were TumorSphere formation assay Cells were placed in 6-well ultralow attachment plates (Corning Inc.) at a density of 1,000 cells/mL in tumorsphere culture medium(Invitrogen, Carlsbad, CA, USA) supplemented with DMEM with 1% N2 supplement, 2% B27 supplement, and 100ng/mL epidermal growth factor at 37°C in a humidified atmosphere of 95% air and 5% CO2. These cells were then treated with different concentrations of SFN at the same time. Primary spheroids were collected following 14 days of culture, and tumorspheres were measured using an inverted microscope system (magnification, Eclipse Ti-s, Nikon, Tokyo, Japan).

Reverse transcription and qPCR analysis
The total RNA was extracted from the cancer cells and cancer stem cells with Trizol reagents. cDNA was synthesized from 1μg of mRNA with a high capacity cDNA reverse

Statistical Analysis
Statistical analysis was performed using SPSS16.0 software. Data presented were mean ± SD from three different experiments. Statistical significance between different groups was determined using Students t-test. A value of P<0.05 was considered statistically significant.

SFN inhibits the growth of lung cancer cells
To explore the role of SFN in lung cancer cells. We first detected cell viability through the MTT assay. The result showed that SFN could inhibit the growth of lung cancer A549 and H460 cells, which was clearly indicated by a declined cell viability trend in a dosedependent manner following treatment with different concentrations of SFN (Fig. 1A).
Electron microscope also shows a significant decrease in the density of A549 and H460 cells following treatments with 8μmol/L SFN for 48h (Fig. 1B). Moreover, the data shows that the number of cells in S phase and G2M phase decreased significantly after treatment with SFN (Fig. 1C).
SFN inhibits the growth of lung cancer stem cells 9 To investigate whether SFN sensitized to lung cancer stem cell, we first isolated CD133+ cells from the human lung cancer A549 and H460 cells by MACS. Compared with 2.8% and 2.2% after isolation CD133+ cells accounted for 96.4% and 97.8% in A549 and H460 cells separately (Fig. 2Aand 2B).The CD133+cells can form a colony in the tumorsphere culture medium. Pluripotency maintaining transcription factor SOX2 plays an important role in maintaining the distinct characteristics of cells in tumors. In this study, we observed that the expression of SOX2 in CD133 positive cells was higher than that in CD133 negative cells (Fig. 2C).
In sphere formation assay, the A549/CD133+ and H460/CD133+ cells were treated with 0, 4, 8 and 12μmol/L SFN. At the end of 14 days, the total number of spheres and the volume of tumorspheres were measured, and the volume of tumorsphere trend showed a gradual decrease with increasing dose of SFN (Fig. 2D). These results showed that SFN can effectively inhibit the proliferation of lung cancer stem cells.
High expression of SHH and PHC3 signaling pathways in lung cancer stem cells It has been reported that the SHH and PHC3 signaling pathways play an important role in the control of stem cell growth [7,14]. However, the role of the SHH and PHC3 signaling pathways in lung CSCs is uncertain. As shown in Fig (Fig. 5AD). When the Shh gene was silenced successfully, the ability of shh-siRNA transfected cells to form tumorsphere became lower than that of the control group (Fig. 5BE). Further research revealed that the inhibitory effect of SFN on cell proliferation still exists in Shh-siRNA cells. In the cell proliferation assay, the cell viability also showed a gradual decrease with increasing dose of SFN both in the siRNA-SHH group and the NT-siRNA group. SFN combined with Shh gene silencing had a greater effect on the proliferation of lung cancer cells (Fig. 5C and 5F).As no prior report on the relationship between SHH and PHC3 in lung cancer exists, we employed a further study expression of PHC3 in shh-siRNA transfected cells. Interestingly, PHC3 expression level showed a significant decrease in shh-siRNA cells compared with the control(as shown in Fig.5AD).

Discussion
A series of scientific studies have shown that SFN can induce apoptosis in a variety of human cancers [22], leading to cell cycle arrest and inhibition of malignant growth. SFN is an isothiocyanate (ITC) extracted from cruciferous vegetables. In our study, we observed that SFN can inhibit the activity of lung cancer cells in vitro.
CD133 is an important marker of cancer stem cells [23,24]. Therefore, we separated lung cancer stem cells which express CD133-positive from the human lung cancer A549 and A460 cells. A series of research studies [25,26] have reported that pluripotency maintaining transcription factor SOX2 plays an important role in maintaining the stem characteristics of cells in tumors. Here, we demonstrate that SOX2 is highly expressed in CD133-positive lung cancer cells. Therefore, CD133 positive cells possess the characteristics of lung cancer stem cells. To investigate whether SFN affects lung cancer stem cells, we performed the sphere formation assay. We observed that the volume and the number of tumorspheres were gradually decreased with the increasing dose of SFN.
This result indicates that SFN has the potential to inhibit the proliferation of lung cancer stem cells. However, the mechanism of the phenomenon still needs to be elucidated.
In recent years, several studies have reported that the SHH signaling pathway plays a critical role in the development and progression of lung cancers [27][28][29]. It has also been reported that the SHH signaling pathway could regulate self-renewal and proliferation of cancer stem cells and increase tumor invasiveness [30]. In this study, we have demonstrated that the SHH signaling pathway was highly upregulated in lung CSCs, suggesting that the hyperactive SHH signaling may regulate the expression of stemness genes in lung CSCs and play important roles in the cell proliferation and progression of lung CSCs. Furthermore, when the Shh gene was silenced successfully, the ability of shh-siRNA transfected cells to form tumor sphere was decreased as compared to that of the control group. Therefore, we speculate that the SHH signal pathway plays an important role in the proliferation of lung cancer cells.
To explore whether SFN inhibits the proliferation of lung cancer stem cells through the SHH signaling pathway, we further investigated the expression of key components in the SHH signaling pathway in cells which were treated by SFN. The results showed that SFN can obviously reduce the mRNA and protein expression of Shh Smo and Gli1 in CD133positive cells as compared to CD133-negative cells. In this study, we also observed that this effect of SFN-induced cell viability inhibition also occurred in A549 andH460 cells in which the Shh gene was silenced. Furthermore, the inhibitory effect of SFN on cell proliferation was stronger in the siRNA-SHH group than in the control group. Therefore, targeted Shh gene therapy is inferred to be a new way to treat lung cancer.
PHC3 is one of the members of PRCs, which have essential roles in early embryonic development and have been implicated in embryonic stem cell pluripotency [31,32]. It has been reported that PHC1 is important in regulating stem cells [33,34]. Other previous studies have shown that PcG complexes control cellular proliferation and favor tumorigenesis [35,36].AM Deshpande [37] reported that PHC3 expression was abnormal in osteosarcoma. In this study, we also investigated whether PHC3 like PHC1 plays the same role in regulating stem cells. Our data indicated that both the mRNA and protein expressions of PHC3 were also markedly increased in human lung CSCs. Further research 13 also showed that SFN inhibited the mRNA and protein expressions of PHC3 in CD133positive cells as compared to CD133-negative cells. Moreover, PHC3 presented the same expression pattern as SHH signaling in lung cancer. However, there is no evidence to support the interaction between SHH and PHC3. Therefore, we investigated the protein expression of PHC3 in Shh silenced cells. Here, we demonstrated that knockdown of shh suppressed PHC3 protein expression in A549 and H460 cells.

Conclusion
This study provides evidence that SFN can serve as a potent anticancer agent, inhibit the proliferation of lung cancer cells and kill the lung cancer stem cells. Furthermore, this study, for the first time, demonstrates that the SHH and PHC3 signaling pathways work together in lung cancer stem cells and aberrant activation of these signals promotes tumorigenesis and progression of lung cancer. These findings suggested that SFN could be exploited in a novel therapeutic avenue for lung cancer treatment by regulating the SHH and PHC3 signaling pathways. The funding agency was not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.