Overactivation of Wnt signaling is a hallmark of colorectal cancer (CRC). The Wnt pathway is a key regulator of both the early and the later, more invasive, stages of CRC development. In the normal intestine and colon, Wnt signaling controls the homeostasis of intestinal stem cells (ISCs) that fuel, via proliferation, upward movement of progeny cells from the crypt bottom toward the villus and differentiation into all cell types that constitute the intestine. Studies in recent years suggested that cancer stem cells (CSCs), similar to ISCs of the crypts, consist of a small subpopulation of the tumor and are responsible for the initiation and progression of the disease. Although various ISC signature genes were also identified as CRC markers and some of these genes were even demonstrated to have a direct functional role in CRC development, the origin of CSCs and their contribution to cancer progression is still debated. Here, we describe studies supporting a relationship between Wnt-regulated CSCs and the progression of CRC.
Changes in cell adhesion and motility are considered key elements in determining the development of invasive and metastatic tumors. Co-opting the epithelial-to-mesenchymal transition (EMT) process, which is known to occur during embryonic development, and the associated changes in cell adhesion properties in cancer cells are considered major routes for tumor progression. More recent in vivo studies in tumor tissues and circulating tumor cell clusters suggest a stepwise EMT process rather than an “all-or-none” transition during tumor progression. In this commentary, we addressed the molecular mechanisms underlying the changes in cell adhesion and motility and adhesion-mediated signaling and their relationships to the partial EMT states and the acquisition of stemness traits by cancer cells.
FSH and Testosterone (T) regulate spermatogenesis via testicular Sertoli cells (Sc), which bear receptors for these hormones. Despite sufficient circulating levels of FSH and T postnatally, predominant appearance of spermatogonia B and spermatocytes is not discernible until 11 and 18 days of postnatal age, respectively, in rat testes. In an attempt to explore the underlying causes, we cultured Sc from neonatal (5- and 9-day-old) and prepubertal (12- and 19-day-old) rat testes and compared the status of FSH receptor (FSH-R) and androgen receptor (AR) signaling. Protein and mRNA levels of FSH-R and AR remained uniform in cultured Sc from all age groups. Androgen binding ability of AR was similar, and T-induced nuclear localization of AR was discernible in Sc from all age groups. Binding of FSH to FSH-R, subsequent production of cAMP, and mRNA of stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF), known to be essential for the robust differentiation of repopulating spermatogonia, were significantly augmented in prepubertal Sc compared with those in neonatal Sc. However, treatment of neonatal Sc with cholera toxin or forskolin, which stimulate cAMP production bypassing FSH-R, demonstrated a concomitant rise in SCF and GDNF mRNA expression, which was similar to the FSH-mediated rise observed in prepubertal Sc. These observations suggested that, during prepubertal Sc maturation, the ability of FSH-R to respond to FSH is significantly augmented and is associated with the robust differentiation of repopulating spermatogonia, and such a switch in Sc from FSH-resistant to FSH-responsive mode during prepubertal development may underlie the initiation of robust spermatogenesis.
Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.
Hyperactivation of Wnt/β-catenin target genes is considered a key step in human colorectal cancer (CRC) development. We previously identified the immunoglobulin-like cell adhesion receptor L1 as a target gene of β-catenin/TCF transactivation that is localized at the invasive edge of CRC tissue. Using gene arrays, we discovered a number of downstream target genes and signaling pathways conferred by L1 overexpression during colon cancer progression. Here, we have used a proteomic approach to identify proteins in the secretome of L1-overexpressing CRC cells and studied the role of the increase in the aspartate protease cathepsin D (CTSD) in L1-mediated colon cancer development. We found that in addition to the increase in CTSD in the secretome, the RNA and protein levels of CTSD were also induced by L1 in CRC cells. CTSD overexpression resulted in elevated proliferation under stress and increased motility, tumorigenesis and liver metastasis, although to a lesser extent than after L1-transfection. The suppression of endogenous CTSD in L1-expressing cells blocked the increase in the proliferative, motile, tumorigenic and metastatic ability of CRC cells. Enhancing Wnt/β-catenin signaling by the inhibition of GSK3β resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/β-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in epithelial cells and in the stromal compartment at the more invasive areas of the tumor, but not in the normal mucosa, indicating that CTSD plays an essential role in CRC progression.
FSH acts via testicular Sertoli cells (Sc) bearing FSH receptor (FSH-R) for regulating male fertility. Despite an adult-like FSH milieu in infant boys and monkeys, spermatogenesis is not initiated until the onset of puberty. We used infant and pubertal monkey Sc to reveal the molecular basis underlying developmental differences of FSH-R signaling in them. Unlike pubertal Sc, increasing doses of FSH failed to augment cAMP production by infant Sc. The expression of Gαs subunit and Ric8b, which collectively activate adenylyl cyclase (AC) for augmenting cAMP production and gene transcription, were significantly low in infant Sc. However, forskolin, which acts directly on AC bypassing FSH-R, augmented cAMP production and gene transcription uniformly in both infant and pubertal Sc. FSH-induced Gαs mRNA expression was higher in pubertal Sc. However, Gαi-2 expression was down-regulated by FSH in pubertal Sc, unlike infant Sc. FSH failed, but forskolin or 8-Bromoadenosine 3',5'-cyclic monophosphate treatment to infant Sc significantly augmented the expression of transferrin, androgen binding protein, inhibin-β-B, stem cell factor, and glial-derived neurotropic factor, which are usually up-regulated by FSH in pubertal Sc during spermatogenic onset. This suggested that lack of FSH mediated down-regulation of Gαi-2 expression and limited expression of Gαs subunit as well as Ric8b may underlie limited FSH responsiveness of Sc during infancy. This study also divulged that intracellular signaling events downstream of FSH-R are in place and can be activated exogenously in infant Sc. Additionally, this information may help in the proper diagnosis and treatment of infertile individuals having abnormal G protein-coupled FSH-R.
Testicular Sertoli cells make a niche for the division and differentiation of germ cells. Sertoli cells respond to increased follicle-stimulating hormone (FSH) and testosterone (T) levels at the onset of puberty by producing paracrine factors which affect germ cells and trigger robust onset of spermatogenesis. Such paracrine support to germ cells is absent during infancy, despite Sertoli cells being exposed to high FSH and T within the infant testis. This situation is similar to certain cases of male idiopathic infertility where post-pubertal Sertoli cells fail to support germ cell division and differentiation in spite of endogenous or exogenous hormonal support. Defective Sertoli cells in such individuals may fail to express the full complement of their paracrine repertoire. Identification and supplementation with such factors may overcome Sertoli cells deficiencies and help trigger quantitatively and qualitatively normal differentiation of germ cells. To this end, we compared the transcriptome of FSH- and T-treated infant and pubertal monkey Sertoli cells by DNA microarray. Expression of Wnt3, a morphogen of the Wnt/β-catenin pathway, was higher in pubertal Sertoli cells relative to infant Sertoli cells. Transgenic mice were generated by us in which Wnt3 expression was curtailed specifically in post-pubertal Sertoli cells by shRNA. Subfertility and oligozoospermia were noticed in such animals with low Wnt3 expression in post-pubertal Sertoli cells along with diminished expression of Connexin43, a gap-junctional molecule essential for germ cell development. We report that the FSH- and T-targetedf Wnt3 governs Sertoli cell-mediated regulation of spermatogenesis and hence is crucial for fertility.
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