Thai-Yen Ling

Cellular growth inhibition by IGFBP-3 and TGF-β1 requires LRP-1

The type V TGF‐β receptor (TβR‐V)/IGFBP‐3 receptor mediates the IGF‐independent growth inhibition induced by IGFBP‐3. It also mediates the growth inhibitory response to TGF‐β1 in concert with other TGF‐β receptor types, and its loss may contribute to the malignant phenotype of human carcinoma cells. Here we demonstrate that TβR‐V is identical to LRP‐1/α2M receptor as shown by MALDI‐TOF analysis of tryptic peptides of TβR‐V purified from bovine liver. In addition, 125I‐IGFBP‐3 affinity‐labeled TβR‐V in Mv1Lu cells is immunoprecipitated by antibodies to LRP‐1 and TβR‐V. RAP, an LRP‐1 antagonist, inhibits binding of 125I‐TGF‐β1 and 125I‐IGFBP‐3 to Tβ R‐V and diminishes IGFBP‐3‐induced growth inhibition in Mv1Lu cells. Absent or low levels of LRP‐1, as with TβR‐V, have been linked to the malignant phenotype of carcinoma cells. Mutagenized Mv1Lu cells selected for reduced expression of LRP‐1 have an attenuated growth inhibitory response to TGF‐β1 and IGFBP‐3. LRP‐1‐deficient mouse embryonic fibroblasts lack a growth inhibitory response to TGF‐β1 and IGFBP‐3. On the other hand, stable transfection of H1299 human lung carcinoma cells with LRP‐1 cDNA restores the growth inhibitory response. These results suggest that the LRP‐1/TβR‐V/IGFBP‐3 receptor is required for the growth inhibitory response to IGFBP‐3 and TGF‐β1.—Huang, S. S., Ling, T.‐Y., Tseng, W.‐F., Huang, Y.‐H., Tang, F.‐M., Leal, S. M., Huang, J. S. Cellular growth inhibition by IGFBP‐3 and TGF‐β1 requires LRP‐1. FASEB J. 17, 2068–2081 (2003)

Cancer-associated fibroblasts regulate the plasticity of lung cancer stemness via paracrine signalling

Cancer stem cells (CSCs) are a promising target for treating cancer, yet how CSC plasticity is maintained in vivo is unclear and is difficult to study in vitro. Here we establish a sustainable primary culture of Oct3/4(+)/Nanog(+) lung CSCs fed with CD90(+) cancer-associated fibroblasts (CAFs) to further advance our knowledge of preserving stem cells in the tumour microenvironment. Using transcriptomics we identify the paracrine network by which CAFs enrich CSCs through de-differentiation and reacquisition of stem cell-like properties. Specifically, we find that IGF1R signalling activation in cancer cells in the presence of CAFs expressing IGF-II can induce Nanog expression and promote stemness. Moreover, this paracrine signalling predicts overall and relapse-free survival in stage I non-small cell lung cancer (NSCLC) patients. IGF-II/IGF1R signalling blockade inhibits Nanog expression and attenuates cancer stem cell features. Our data demonstrate that CAFs constitute a supporting niche for cancer stemness, and targeting this paracrine signalling may present a new therapeutic strategy for NSCLC.

Synthetic TGF-β antagonist accelerates wound healing and reduces scarring

Wound healing consists of re‐epithelialization, contraction and formation of granulation and scar tissue. TGF‐β is involved in these events, but its exact roles are not well understood. Here we demonstrate that topical application of a synthetic TGF‐β antagonist accelerates re‐epithelialization in pig burn wounds (100% re‐epithelialization in antagonist‐treated wounds vs. ~ 70% reepithelialization in control wounds on postburn day 26) and reduces wound contraction and scarring in standard pig skin burn, pig skin excision and rabbit skin excision wounds. These results support the distinct roles of TGF‐β in the complex process of wound healing and demonstrate the feasibility of manipulating wound healing by TGF‐β antagonist.

Expression of the pluripotent transcription factor OCT4 promotes cell migration in endometriosis.

OBJECTIVE To identify the impact of the pluripotent transcription factor OCT4 in endometrial cell migration and endometriosis. DESIGN The OCT4 expression and cell migration study. SETTING Research institution and reproductive medical clinic. PATIENT(S) Nine subjects with normal endometrium, 3 subjects with normal myometrium, 36 patients with hyperplastic endometrium, and 58 patients with endometriosis. INTERVENTION(S) The expression of OCT4 messenger RNA in normal endometrium, normal myometrium, hyperplastic endometrium, and ectopic endometriotic tissues was analyzed using reverse transcription and quantitative real-time polymerase chain reaction (PCR). The effect of OCT4 expression on the migration activity of the endometrial cells was examined. MAIN OUTCOME MEASURE(S) Reverse transcription and quantitative real-time PCR, Western blotting, and wound closure and transwell assays. RESULT(S) The expression of OCT4 and NANOG messenger RNA was significantly higher in ectopic endometriotic tissues, compared with that of the normal endometrium, the normal myometrium, and the hyperplastic endometrium. The level of OCT4 messenger RNA in endometriotic tissues was positively correlated with the expression of genes associated with cell migration. Overexpression of the OCT4 protein in primary human endometriotic stromal cells and human RL95-2 and HEC1A endometrial carcinoma cell lines resulted in decreased levels of E-CADHERIN, the increased expression of the VIMENTIN, TWIST, and SLUG proteins, and an increase in the migration activity of endometrial cells in transwell and wound closure assays. CONCLUSION(S) The transcription of the OCT4 gene is significantly up-regulated in human ectopic endometriotic tissues. The expression of OCT4 may contribute to the pathology of ectopic endometrial growth by stimulating the migration activity of endometrial cells.

Epithelial Notch signaling regulates lung alveolar morphogenesis and airway epithelial integrity

Significance Formation of the gas-exchange region of the lung occurs largely postnatally through a process called alveologenesis. Alveolar abnormalities are a hallmark of neonatal and adult chronic lung diseases. Here we report that disruption of Notch signaling in mice, particularly by Notch2, results in abnormal enlargement of the alveolar spaces reminiscent of that seen in chronic lung diseases. We provide evidence that Notch is crucial to mediate cross-talk between different cell layers, including signals such as PDGF for formation of the alveoli and maintenance of the integrity of the conducting airways. Abnormal enlargement of the alveolar spaces is a hallmark of conditions such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. Notch signaling is crucial for differentiation and regeneration and repair of the airway epithelium. However, how Notch influences the alveolar compartment and integrates this process with airway development remains little understood. Here we report a prominent role of Notch signaling in the epithelial–mesenchymal interactions that lead to alveolar formation in the developing lung. We found that alveolar type II cells are major sites of Notch2 activation and show by Notch2-specific epithelial deletion (Notch2cNull) a unique contribution of this receptor to alveologenesis. Epithelial Notch2 was required for type II cell induction of the PDGF-A ligand and subsequent paracrine activation of PDGF receptor-α signaling in alveolar myofibroblast progenitors. Moreover, Notch2 was crucial in maintaining the integrity of the epithelial and smooth muscle layers of the distal conducting airways. Our data suggest that epithelial Notch signaling regulates multiple aspects of postnatal development in the distal lung and may represent a potential target for intervention in pulmonary diseases.

Chemotherapeutic sensitivity of testicular germ cell tumors under hypoxic conditions is negatively regulated by SENP1-controlled sumoylation of OCT4.

Testicular germ cell tumors (TGCT) generally respond well to chemotherapy, but tumors that express low levels of the transcription factor OCT4 are associated with chemoresistance and poor prognosis. Hypoxia is known to induce drug resistance in TGCTs; however, the mechanistic basis for reduced expression of OCT4 and drug resistance is unclear. Here we show that hypoxia reduces OCT4 levels and increases the resistance of embryonal carcinoma (EC) cells to cisplatin and bleomycin. Furthermore, we show that the loss of OCT4 expression under hypoxia can be triggered by sumoylation, which was regulated by SUMO1 and the SUMO1 peptidase SENP1. Under hypoxic conditions, overexpression of SUMO1gg (the active form of SUMO1) not only increased the level of sumoylated OCT4 (Su-OCT4), but also decreased the stability of OCT4 protein. In addition, overexpression of SENP1 reduced the Su-OCT4 level induced by SUMO1gg overexpression, thereby maintaining OCT4 levels and enhancing chemosensitivity. Mechanistic investigations revealed that OCT4 sumoylation occurred at K123, as overexpression of an OCT4-K123R mutant effectively reduced the level of Su-OCT4 under hypoxic conditions. Taken together, our results showed that hypoxia reduces OCT4 expression levels in ECs to increase drug resistance and that these effects could be countered to ablate the suppressive effects of hypoxia on chemosensitivity. Our findings also highlight SENP1 as a potential therapeutic target for drug resistant TGCTs.

Sustained release of adipose-derived stem cells by thermosensitive chitosan/gelatin hydrogel for therapeutic angiogenesis

Adipose-derived stem cells (ASCs) secrete several angiogenic growth factors and can be applied to treat ischemic tissue. However, transplantation of dissociated ASCs has frequently resulted in rapid cell death. Therefore, we aimed to develop a thermosensitive chitosan/gelatin hydrogel that is capable of ASC sustained release for therapeutic angiogenesis. By blending gelatin in the chitosan thermosensitive hydrogel, we significantly enhanced the viability of the encapsulated ASCs. During in vitro culturing, the gradual degradation of gelatin led to sustained release of ASCs from the chitosan/gelatin hydrogel. In vitro wound healing assays revealed significantly faster cell migration by co-culturing fibroblasts with ASCs encapsulated in chitosan/gelatin hydrogel compared to pure chitosan hydrogels. Additionally, significantly higher concentrations of vascular endothelial growth factor were found in the supernatant of ASC-encapsulated chitosan/gelatin hydrogels. Co-culturing SVEC4-10 endothelial cells with ASC-encapsulated chitosan/gelatin hydrogels resulted in significantly more tube-like structures, indicating the hydrogels potential in promoting angiogenesis. Chick embryo chorioallantoic membrane assay and mice wound healing model showed significantly higher capillary density after applying ASC-encapsulated chitosan/gelatin hydrogel. Relative to ASC alone or ASC-encapsulated chitosan hydrogel, more ASCs were also found in the wound tissue on post-wounding day 5 after applying ASC-encapsulated chitosan/gelatin hydrogel. Therefore, chitosan/gelatin thermosensitive hydrogels not only maintain ASC survival, they also enable sustained release of ASCs for therapeutic angiogenesis applications, thereby exhibiting great clinical potential in treating ischemic diseases. STATEMENT OF SIGNIFICANCE Adipose-derived stem cells (ASCs) exhibit great potential to treat ischemic diseases. However, poor delivery methods lead to low cellular survival or dispersal of cells from target sites. In this study, we developed a thermosensitive chitosan/gelatin hydrogel that not only enhances the viability of the encapsulated ASCs, the gradual degradation of gelatin also result in a more porous architecture, leading to sustained release of ASCs from the hydrogel. ASC-encapsulated hydrogel enhanced in vitro wound healing of fibroblasts and tube formation of endothelial cells. It also promoted in vivo angiogenesis in a chick embryo chorioallantoic membrane assay and a mice wound model. Therefore, chitosan/gelatin hydrogel represents an effective delivery system that allows for controlled release of viable ASCs for therapeutic angiogenesis.

Fatty acids modulate transforming growth factor-β activity and plasma clearance

The activity and plasma clearance of transforming growth factor (TGF)‐β are known to be regulated by activated α2‐macroglobulin (α2M*). This has been implicated in pathophysiological processes, but no small molecule compounds have been reported to modulate TGF‐β activity by affecting the interaction of TGF‐β and α2M*. Here, we demonstrate that fatty acids are capable of inhibiting complex formation of TGF‐β isoforms and α2M* as demonstrated by nondenaturing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. This is dependent on carbon chain length (C20, C18, C16, C14 > C12 > C10), degree of unsaturation (polyunsaturated > saturated), and TGF‐β isoforms (TGF‐β1 > TGF‐β2 > TGF‐β3). Arachidonic acid, which is one of the most potent inhibitors, is also capable of dissociating TGF‐β‐α2M* complexes, but higher concentrations are required. Arachidonic acid appears to inhibit TGF‐β‐α2M* complex formation by binding specifically to α2M* as demonstrated by gel filtration chromatography. Arachidonic acid reverses the inhibitory effect of α2M* on TGF‐β binding, TGF‐β‐induced growth inhibition, and TGF‐β‐induced transcriptional activation in mink lung epithelial cells and affects plasma clearance of TGF‐β‐α2M* complexes in mice. These results show that fatty acids are effective modulators of TGF‐β activity and plasma clearance and may be useful in treating human diseases through their effects on the interaction of TGF‐β and α2M*.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Enhancement role of host 12/15-lipoxygenase in melanoma progression

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.