Breast cancer is a most common cancer in women worldwide. Although the prognosis of the primary tumors has improved significantly with new therapeutic techniques in recent years, the prognosis of metastatic breast cancer remains a major challenge in clinical work. Tumor metastasis is a complex biological process, involving many genes and biomolecules. Until now, the understanding of breast cancer metastasis is still incomplete. It is urgent to fully understand the biological basis of metastasis to improve the long-term survival of breast cancer patients.
The pseudopodium, which is mainly composed of lamellipodium and filopodium, participates in tumor metastasis. The formation of pseudopodium depends on microfilaments[5– 6], which are regulated by Rho family small GTPases. The Rac subfamily of Rho GTPases activates the front edge of cells. A representative member of Rac family is Rac1, which regulates the polymerization of the branched microfilaments, and thus plays a key role in the formation and function of lamellipodiums. In our earlier study, Wnt5a, a nonclassical Wnt signal promotes the migration of MCF-7 breast cancer cells through Rac1. However, migration involves many signaling pathways, so exploring the molecular mechanisms of migration is critical for precise selection of therapeutic targets during clinical treatment. An appropriate combination of multiple signaling pathway inhibitors can improve the efficacy of treatment and reduce the incidence of drug resistance.
It has been demonstrated that Sonic Hedgehog (Shh) signal is required for the growth and proliferation of a variety of tumors[9– 10]. Shh is an essential morphogenic and mitogenic factor in embryonic development and postnatal physiological processes. Shh signaling is initiated by the binding of Shh ligand to the 12-pass transmembrane receptor Patched-1 (Ptch1), which attenuates the inhibition of Ptch1 on Smoothened (Smo), a G-protein coupled receptor. Activated Smo turns on the downstream transcription program orchestrated by three transcription factors: Glioma-associated oncogene homolog 1, 2, and 3 (Gli1, Gli2 and Gli3). Mouse mammary cancer model studies have found the Shh-dependent mechanisms in mammary gland tumor formation and development. Inhibition of Shh signaling reduces the survival of tumor cells and the abnormal stimulation of basal cells. However, it is not clear whether Shh contributes to metastasis of breast cancer.
In this study, we found that in MCF-7 breast cancer cells, Shh signal regulates the activation of Rac1, thereby affecting cell migration independent of Wnt5a, which called for further exploration of the potential crosstalk between two important signaling pathways of Wnt5a and Shh. It is also suggested that key molecules of the Shh signaling can be used as drug targets and combined with other drugs, to contribute to clinical breast cancer treatment.
It has been reported that the expression of Shh in breast cancers is higher than in normal mammary tissues. TCGA database searching and UALCAN website analysis showed that Shh expression in tissues of luminal breast cancer, Her2 positive breast cancer and triple negative breast cancer was higher than that in normal breast tissues (Fig. 1A).
Further, we selected patient specimens with complete clinical records and pathological data for immunohistochemistry (IHC) from Department of Pathology of Jiangsu Province Hospital. The slices were divided into 6 groups according to relevant biomarkers to rule out potential inter-group differences (Table 1). IHC results showed that the expression of Shh was significantly increased in breast cancer compared to that in normal breast tissues (Fig. 1B), which is consistent with TCGA analysis.
Diagnosis Case Shha P Negative [n (%)] Positive [n (%)] Normal (n=20) - 20 18 (90.0) 2 (10.0) < 0.01 ER(–), PR(–), Her2(–) 19 0 (0.0) 19 (100.0) ER(+), PR(+), Her2(–) 47 0 (0.0) 47 (100.0) Tumor (n=100) ER(–), PR(–), Her2(+) 16 0 (0.0) 16 (100.0) ER(+), PR(+), Her2(+) 14 0 (0.0) 14 (100.0) ER(+/–), PR(–/+), Her2(+/–)b 4 0 (0.0) 4 (100.0) The differences between the two groups were evaluated by χ2 tests.
aImmunoreactivity score of Shh: (–) as negative; (+)~(+++) as positive.
bER(+/–), PR(–/+), Her2(+/–): ER(+), PR(–), Her2(+); ER(–), PR(+), Her2(+); ER(+), PR(–), Her2(–); ER(–), PR(+), Her2(–).
Table 1. Association between Shh and ER, PR, Her2 expression in breast tissues
To understand the function of Shh in breast cancer, we treated MCF-7 breast cancer cells with Shh conditioned medium. The migration ability of MCF-7 cells was examined by wound healing assay and transwell migration assay. MCF-7 cells treated Shh ligand closed the wound faster than control cells (Fig. 2A). They showed significantly enhanced migration ability in Boyden chambers without collagen coat as well (Fig. 2B). These results indicated that Shh promotes the migration of MCF-7 cells.
Since Smo and Gli1 are both positive effectors of Shh signaling, we speculated if Smo and Gli1 could promote migration of MCF-7 cells. We incubated MCF-7 cells with different doses of Smo agonist (SAG), Smo inhibitor (vismodegib, GDC-0449) or Gli1 inhibitor (GANT-61), or transfected MCF-7 cells with HA-Gli1 plasmids, respectively. Those cells then underwent wound healing assay (Fig. 2C). The results showed that Smo activation and exogenous Gli1 promoted the MCF-7 migration, while the inhibition of Smo and Gli1 impeded it. We also performed transwell migration assay and got the consistent conclusion (Fig. 2D). These results identified that Shh signal promotes the migration of MCF-7 breast cancer cells through Smo and Gli1.
Cytoskeleton drives shape changes during cell migration. Therefore, we incubated MCF-7 cells with control and Shh ligand, and then stained F-actin with FITC-phalliodin. Fluorescent staining indicated that Shh increased the quantity of stress fibers and lamellipodium of MCF-7 breast cancer cells (Fig. 3A). Rac1 was known to be involved in the information of lamellipodium, so we speculated that Rac1 activity might regulate the stimulation of cell migration by Shh in MCF-7 breast cancer cells.
It was demonstrated in our earlier study that Rac1 regulated MCF-7 breast cancer cell migration as downstream of Wnt5a pathway. According to the results above, a GST-pulldown assay (Fig. 3B) was proceeded to investigate Rac1 activation under Shh stimulation. Active Rac1 could be pulled down by GST fused Pak1 protein-binding domain (PBD) (Fig. 3B). We treated MCF-7 cells with Shh ligand for different time periods. Immunoblotting showed increased active Rac1 was pulled down from Shh treated cells. Activation of Rac1 peaked at 15 minutes of Shh treatment (Fig. 3C).
We further investigated the function of Smo and Gli1 on Rac1 activation in the same way. The results showed that SAG treatment and ectopic expression of HA-Gli1 significantly promoted Rac1 activation in MCF-7 cells, whereas GDC-0449 and GANT-61 had opposite effects (Fig. 3C–G). It indicated that Smo and Gli1 both positively regulated Rac1 activity.
To further verify the results above, we used siRNA to interfere Rac1 expression, or Rac GTPase inhibitor EHop-016 to block Rac1 activation, respectively. EHop-016 could significantly reduce the migration and the Rac1 activation of MCF-7 cells (Supplementary Fig. 1, available online), which was consistent with si-Rac1.
We found that Smo and Gli1 promoted cell migration and Rac1 activation of MCF-7 cells. However, SAG failed to promote the migration of Rac1-knockdown MCF-7 in wound healing assay and transwell migration assay. And HA-Gli1failed as well (Fig. 4A and 4B). EHop-016 completely blocked the effect of SAG and exogenous Gli1 on Rac1 activation in MCF-7 cells (Fig. 4C and 4D).
Figure 4. Inhibition of Rac1 activation blocks the effect of Shh on the migration of MCF-7 breast cancer cells.
So far, we confirmed that Shh could promote the migration of MCF-7 breast cancer cells by up-regulating Rac1 activation.
Our published study showed that Wnt5a controled the activation of Rac1 on MCF-7 cell migration through Dvl2 and Rab35. Since Shh could also regulate Rac1 activation, we were curious about the crosstalk between Shh and Wnt5a pathway in cell migration. In search of an answer, we knocked down Dvl2 and Rab35 respectively in MCF-7 cells by siRNA, then treated the cells with SAG or exogenously expressed Gli1. The results showed that the siRNA reduced the migration rate (Fig. 5A and 5B, 5E and 5F) and Rac1 activation (Fig. 5C and 5D, 5G and 5H), which were both reversed by SAG and HA-Gli1. These results suggested that Shh ddi not depend on the Wnt5a-Dvl2-Rab35 axis to regulate Rac1 activation.
Figure 5. Shh signaling regulates MCF-7 breast cancer cell migration and Rac1 activation independent of Wnt5a.
In summary, we present the evidence that both Wnt5a and Shh promote breast cancer cell migration via Rac1 (Fig. 6). These findings highlight the presence of a potential molecular mechanism of resistance, which may represent a rational molecular target for combination medication in breast cancer.