EphB6 deficiency in intestinal neurons promotes tumor growth in colorectal cancer by neurotransmitter GABA signaling.

EphB6 belongs to the receptor tyrosine kinase, whose low expression is associated with shorter survival of colorectal cancer (CRC) patients. But the role and mechanism of EphB6 in the progression of CRC need further study. In addition, EphB6 was mainly expressed in intestinal neurons. But how EphB6 is involved in functions of intestinal neurons has not been known. In our study, we constructed a mouse xenograft model of CRC by injecting CMT93 cells into the rectum of EphB6-deficient mice. We found that the deletion of EphB6 in mice promoted tumor growth of CMT93 cells in a xenograft model of CRC, which was independent of changes in the gut microbiota. Interestingly, inhibition of intestinal neurons by injecting botulinum toxin A into rectum of EphB6-deficient mice could eliminate the promotive effect of EphB6 deficiency on tumor growth in the xenograft model of CRC. Mechanically, the deletion of EphB6 in mice promoted the tumor growth in CRC by increasing GABA in the tumor microenvironment. Furthermore, EphB6 deficiency in mice increased the expression of synaptosomal-associated protein 25 in the intestinal myenteric plexus, which mediated the release of GABA. Our study concluded that EphB6 knockout in mice promotes tumor growth of CMT93 cells in a xenograft model of CRC by modulating GABA release. Our study found a new regulating mechanism of EphB6 on the tumor progression in CRC that is dependent on intestinal neurons.

Prognostic value of delta-like protein 3 combined with thyroid transcription factor-1 in small-cell lung cancer.

Rovalpituzumab tesirine is a promising delta-like protein 3 (DLL3)-targeted antibody-drug conjugate for the treatment of small-cell lung cancer (SCLC). Thyroid transcription factor-1 (TTF-1) and DLL3 protein are associated with SCLC, and may be used to identify patients, who respond to the DLL3-targeted therapy. However, little is known about the expression pattern of the DLL3 protein, and the prognostic value of DLL3 and TTF-1 for SCLC. A total of 335 patients with SCLC were identified, including 11 patients with paired biopsy of primary site and lobectomy specimens, and 37 patients with paired specimens of primary and metastatic site. The DLL3 expression levels of individuals were evaluated using the anti-DLL3 antibody. No differences in DLL3 expression levels were observed in paired biopsy and lobectomy specimens (P=0.774), and paired primary and metastatic sites (P=0.472). SCLC cases with high DLL3 expression levels were more frequent in male patients (P=0.041), smokers (P=0.023) and patients with positive TTF-1 expression (P=0.006) compared with DLL3-low SCLC. DLL3-high SCLC exhibited worse overall survival compared with DLL3-low SCLC (log-rank test, P=0.007). Patients with TTF-1+ SCLC experienced a significantly worse overall survival compared with patients with TTF-1- SCLC (P<0.001). DLL3-low/TTF-1- was defined as a distinct molecular subgroup of SCLC with optimal prognosis (P<0.001). DLL3-low/TTF-1- was an independent prognostic marker for SCLC (P=0.001). In conclusion, the present study, to the best of our knowledge, provided novel evidence for SCLC intratumoral and intertumoral homogeneity with the identification of DLL3 protein levels. Therefore, it is reliable to use biopsy specimens to evaluate DLL3 expression levels for identification of patients who may benefit from DLL3-targeted therapy. In addition, DLL3 and TTF-1 are two protein markers with potential clinical value in risk stratification for patients with SCLC.

A signature for induced pluripotent stem cell-associated genes in colorectal cancer.

Genes associated with induced pluripotent stem cells (iPS genes) are several pivotal transcriptional factors, which are used to induce pluripotent stem cells from some adult somatic cells. The roles of these iPS genes and especially the signature for these iPS genes in colorectal cancer (CRC) are still unclear. Overexpressed Oct4 and Lin28 but down-regulated Nanog were found in tumor tissues compared with that in their paired normal counterparts of CRC patients. Interestingly, we found that Oct4, Lin28 and Nanog were highly overexpressed in some patients. And the signature for iPS genes was correlated with tumor site (P = 0.012), lymph node status (P = 0.033), Dukes classification (P = 0.033) of CRC patients. Moreover, an independent public expression profiling data showed signature for the four iPS genes could successfully be used to predict the survival of CRC patients with Dukes stages B and C. Immunofluorescent staining of fresh CRC tissues from patients showed that strong co-expressions of Oct4 and Nanog proteins or Sox2 and Lin28 were present in some CRC cells. Then, CRC cell subclone with four iPS genes overexpression were establish by a mixed retroviral system. We found that iPS genes promote sphere-formation, proliferation, colony formation, migration of human CRC cells in vitro and tumor growth in vivo. Our study first shows the clinical significance of iPS signature in CRC patients.

Snail as a key regulator of PRL-3 gene in colorectal cancer.

The regulators of a key metastasis gene PRL-3 in colorectal cancer (CRC) are still largely unknown. We found three potential binding sites of Snail, a key transcriptional factor involved in the epithelial-mesenchymal transition (EMT), in the region of PRL-3 promoter (located at -642 to -383). Moreover, our results showed that one of the Snail binding sites (located at -624 to -619) was the key element to maintain promoter activity of human PRL-3 gene. The transcriptional activity of PRL-3 promoter was abolished after the Snail binding site (located at -624 to -619) was mutated. Both promoter activity and protein expression of PRL-3 in CRC cell lines could be regulated by Snail. In clinical samples of CRC and metastatic lymph node of CRC, expression of PRL-3 protein was correlated with expression of Snail protein. Functional studies using gene over-expression and knockdown methods indicated that Snail promoted proliferation, cell adhesion and migration of human CRC cells. In SW480 cells with PRL-3 stable knockdown, cell proliferation increased after Snail was up-regulated. Our data first reveal transcriptional factor Snail as a key regulator of PRL-3 in CRC. The link between Snail and PRL-3 suggests a new potential mechanism of Snail contributing to progression and metastasis of CRC.

Stathmin, a new target of PRL-3 identified by proteomic methods, plays a key role in progression and metastasis of colorectal cancer.

To better understand the role of PRL-3 in progression and metastasis of colorectal cancer (CRC), we searched for PRL-3 associated proteins using proteomic methods. We identified 39 PRL-3 associated proteins based on proteomic strategy. Stathmin, a key oncoprotein, was proved to be a new PRL-3 associated protein. Notably, co-immunoprecipitation assays in both endogenous CRC cell lines and CRC tissues indicated that PRL-3 could interact with stathmin. And, both stathmin and PRL-3 contributed to microtubule (MT) destabilization of CRC cells. Moreover, gain-of-function and loss-of-function analyses revealed that stathmin promoted proliferation, cell adhesion, and migration of human CRC cells. Immunohistochemical analysis of 149 colorectal tumor samples showed that overexpression of stathmin was strongly correlated with tumor differentiation (P = 0.035), tumor invasion (P = 0.024), lymph node status (P < 0.001), Dukes classification (P < 0.001), and TNM staging (P < 0.001) of CRC patients. Univariate and multivariate survival analyses further supported that overexpression of stathmin protein was a potential independent poor prognostic factor for CRC. Our results reveal many PRL-3 associated proteins for the first time. The oncoprotein stathmin plays a key role in CRC as a new target of PRL-3. Interaction between PRL-3 and stathmin leads to MT destabilization of CRC cells, which contributes to progression and metastasis of CRC.