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[This corrects the article DOI: 10.1371/journal.pone.0071093.].

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.

Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr(397). However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr(397) were expressed in bone marrow cells and that pFAK Tyr(397) was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr(397) were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr(397) time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b(+) bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.

Continuous theta-burst stimulation (cTBS) is a noninvasive repetitive brain stimulation protocol that suppresses the excitability of the primary motor cortex. It induces cerebral cortical inhibition by increasing inhibitory interneuronal excitability that is associated with increases in gamma-aminobutyric acid (GABA) concentration in the stimulated cortices. cTBS has been applied in the rehabilitation of stroke patients to modulate interhemispheric imbalance. However, the precise mechanisms of cTBS in remote brain areas remain uncertain. We evaluated cTBS-induced GABA level changes in bilateral sensorimotor cortices using GABA-edited magnetic resonance spectroscopy, alternations of motor evoked potentials (MEPs), and resting-state networks (RSNs) using resting-state functional magnetic resonance imaging in 24 healthy right-handed adults (mean age: 34.4 ± 5.0 years). GABA levels in the stimulated left hemisphere significantly increased from baseline (p = 0.013), which was comparable with those of previous reports. GABA levels in the unstimulated right hemisphere showed a trend decrease. cTBS induced a significant decrease in right hand-MEP amplitudes (22.06% ± 43.50%) from baseline (p = 0.026) in accordance with GABA concentrations. However, multiple RSNs, including the default mode and primary motor networks, did not show any obvious differences between pre- and post-stimulus comparisons in the sensorimotor network using the dual regression approach. These results suggest that cTBS simultaneously increases ipsilateral GABA in the stimulated left hemisphere and decreases contralateral GABA in the unstimulated right hemisphere. Neuromodulation following cTBS may be associated with the interhemispheric inhibition because of alterations in GABA levels between the stimulated and unstimulated cortices.