Cancer-associated fibroblasts mediated chemoresistance by a FOXO1/TGFß1 signaling loop in esophageal squamous cell carcinoma.

Previous studies on the mechanisms underlying ESCC (esophageal squamous cell carcinoma) chemoresistance only focused on tumor cells while tumor microenvironment has been completely ignored. Our study aimed to clarify the effect of CAFs (cancer-associated fibroblasts), one major component of tumor microenvironment, on the chemoresistance of ESCC. By primary culture, two pairs of CAFs and matched NFs (normal fibroblasts) were isolated from tumor tissues of ESCC patients and matched normal esophageal epithelial tissues, respectively. The association of CAFs and chemoresistance was assessed in esophageal carcinoma cells, in xenograft tumor models and in clinical specimens of ESCC patients. We found CAFs conferred ESCC cells significant resistance to several common chemotherapeutic drugs including cisplatin, taxol, irinotecan (CPT-11), 5-fluorouracil (5-Fu), carboplatin, docetaxel, pharmorubicin, and vincristine. Mechanism studies revealed that blockage of CAFs-secreted TGFß1 signaling by its receptor TGFßR1 inhibitor LY2157299 significantly reversed the chemoresistance in vitro and in vivo. Furthermore, the crosstalk of CAFs and ESCC cells enhanced the expression and activation of FOXO1, a member of the forkhead transcription factors in the O-box sub-family, inducing TGFß1 expression in an autocrine/paracrine signaling loop. In 130 ESCC patients, the expression of TGFß1 in CAFs was significantly associated with overall survival of patients treated with chemoradiotherapy. Together, our study highlighted TGFß1 expressed in CAFs as an attractive target to reverse tumor chemoresistance, and can be used as an independent prognostic factor of ESCC patients treated with chemoradiotherapy. © 2016 Wiley Periodicals, Inc.

Noncanonical Activin A Signaling in PC12 Cells: A Self-Limiting Feedback Loop.

Activin A (Act A), a member of transforming growth factor-ß superfamily, plays a neuroprotective role in multiple neurological diseases through Act A/Smads signal activation. Traditionally, the up-regulation of Act A gene and extracellular Act A accumulation show the signal activation as a linear pathway. However, one of our discoveries indicated that Act A could lead a loop signaling in ischemic injury. To clarify the characteristic of this loop signaling in a non-pathological state, we up-regulated the expression of Act A, monitored extracellular Act A accumulation and examined the activity of Act A signaling, which was quantified by the expression of phosphorylated Smad3 and the fluorescence intensity of Smad4 in nuclei. The results demonstrated a noncanonical Act A signal loop with self-amplifying property in PC12 cells. Further, it showed self-limiting behavior due to temporary activation and spontaneous attenuation. This periodic behavior of Act A signal loop was found to be regulated by the level of Smad anchor for receptor activation (SARA). Moreover, increased activity of Act A signal loop could promote PC12 cell proliferation and enhance the survival rate of cells to Oxygen-Glucose Deprivation. These practical discoveries will bring new insight on the functional outcome of Act A signaling in neurological diseases by the further understanding: loop signaling.

Inactivation of Nell-1 in Chondrocytes Significantly Impedes Appendicular Skeletogenesis.

NELL-1, an osteoinductive protein, has been shown to regulate skeletal ossification. Interestingly, an interstitial 11p14.1-p15.3 deletion involving the Nell-1 gene was recently reported in a patient with short stature and delayed fontanelle closure. Here we sought to define the role of Nell-1 in endochondral ossification by investigating Nell-1-specific inactivation in Col2α1-expressing cell lineages. Nell-1flox/flox ; Col2α1-Cre+ (Nell-1Col2α1 KO) mice were generated for comprehensive analysis. Nell-1Col2α1 KO mice were born alive but displayed subtle femoral length shortening. At 1 and 3 months postpartum, Nell-1 inactivation resulted in dwarfism and premature osteoporotic phenotypes. Specifically, Nell-1Col2α1 KO femurs and tibias exhibited significantly reduced length, bone mineral density (BMD), bone volume per tissue volume (BV/TV), trabecular number/thickness, cortical volume/thickness/density, and increased trabecular separation. The decreased bone formation rate revealed by dynamic histomorphometry was associated with altered numbers and/or function of osteoblasts and osteoclasts. Furthermore, longitudinal observations by in vivo micro-CT showed delayed and reduced mineralization at secondary ossification centers in mutants. Histologically, reduced staining intensities of Safranin O, Col-2, Col-10, and fewer BrdU-positive chondrocytes were observed in thinner Nell-1Col2α1 KO epiphyseal plates along with altered distribution and weaker expression level of Ihh, Patched-1, PTHrP, and PTHrP receptor. Primary Nell-1Col2α1 KO chondrocytes also exhibited decreased proliferation and differentiation, and its downregulated expression of the Ihh-PTHrP signaling molecules can be partially rescued by exogenous Nell-1 protein. Moreover, intranuclear Gli-1 protein and gene expression of the Gli-1 downstream target genes, Hip-1 and N-Myc, were also significantly decreased with Nell-1 inactivation. Notably, the rescue effects were diminished/reduced with application of Ihh signaling inhibitors, cyclopamine or GANT61. Taken together, these findings suggest that Nell-1 is a pivotal modulator of epiphyseal homeostasis and endochondral ossification. The cumulative chondrocyte-specific Nell-1 inactivation significantly impedes appendicular skeletogenesis resulting in dwarfism and premature osteoporosis through inhibiting Ihh signaling and predominantly altering the Ihh-PTHrP feedback loop. © 2018 American Society for Bone and Mineral Research.