Metastatic spread is an inefficient process which requires generation of supportive microenvironments in which cancer cells can survive, proliferate and escape from immune attack. These niches are induced by systemic and locally produced factors and establish a tumor-supportive and immune suppressive environment which is molecularly and functionally different from the niche at the primary site. Tumor dormancy may result if the niche is not sufficiently supportive/protective. Co-evolution of cancer cells and the surrounding microenvironment creates a large number of such dynamic niches, and we are just beginning to elucidate the complexity of these interactions and their tissue-specific differences. We will discuss exciting possibilities but also challenges which are immanent when trying to target these stromal responses for diagnosis and therapy.
The Wnt/?-catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/?-catenin signalling pathway as a negative regulator of both basal and stress-induced autophagy. Manipulation of ?-catenin expression levels in vitro and in vivo revealed that ?-catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation ?-catenin is selectively degraded via the formation of a ?-catenin-LC3 complex, attenuating ?-catenin/TCF-driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the ?-catenin-LC3 complex is mediated by a W/YXXI/L motif and LC3-interacting region (LIR) in ?-catenin, which is required for interaction with LC3 and non-proteasomal degradation of ?-catenin. Thus, Wnt/?-catenin represses autophagy and p62 expression, while ?-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place ?-catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy.
Cyclooxygenase-2 (COX-2) overexpression in colorectal cancer increases levels of its pro-tumorigenic product prostaglandin E2 (PGE(2)). The recently identified colorectal tumour suppressor 15-prostaglandin dehydrogenase (15-PGDH) catalyses prostaglandin turnover and is downregulated at a very early stage in colorectal tumorigenesis; however, the mechanism responsible remains unclear. As Wnt/?-catenin signalling is also deregulated early in colorectal neoplasia, a study was undertaken to determine whether ?-catenin represses 15-PGDH expression.
Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1?,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.
We designed by docking and synthesized two novel analogues of 1?,25-dihydroxyvitamin D(3) hydroxymethylated at C-26 (2 and 3). The syntheses were carried out by the convergent Wittig-Horner approach via epoxide 12a as a common key intermediate. The antiproliferative and transactivation potency of the compounds was evaluated in colon and breast cancer cell lines. The analogues showed a similar but reduced activity compared to 1,25(OH)(2)D(3). Analogue 3 was more potent than analogue 2, and in some assays it exhibited potency similar to that of the natural ligand.
Aberrant activation of the Wnt/?-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear ?-catenin and the induction of its target genes. Apc(min/+) mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear ?-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear ?-catenin levels in colon cancer in vivo. By appropriate breeding of Apc(min/+) mice and Vdr(+/-) mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apc(min/+)Vdr(-/-) mice had increased nuclear ?-catenin and the tumors reached a larger size than those of Apc(min/+)Vdr(+/+). Both ACF and carcinomas in Apc(min/+)Vdr(-/-) mice showed higher expression of ?-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced ?-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)(2)D(3) to promote the relocation of ?-catenin from the nucleus to the plasma membrane and to inhibit ?-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear ?-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/?-catenin pathway.
An improved synthetic route to 1?,25-dihydroxyvitamin D(3) des-side chain analogues 2?a and 2?b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF-7 breast cancer cells and prodifferentiation activity toward SW480-ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin?D receptor (hVDR) complexed to one of these analogues, 20(17?18)-abeo-1?,25-dihydroxy-22-homo-21-norvitamin D(3) (2?a) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1?,25-dihydroxyvitamin D(3).
Many studies support a protective action of vitamin D against colon cancer. 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts wide gene regulatory effects in human colon cancer cells. We previously reported that 1,25(OH)2D3 increases cytosolic Ca2+ concentration and transiently activates RhoA and its effector the Rho-associated coiled-kinase (ROCK), and later p38MAPK-MSK. We found that the inhibition of ROCK signaling by Y27632 or that of MSK by Ro318220 prevent the formation of epithelioid islands of SW480-ADH cells by 1,25(OH)2D3 and disrupts the adhesive phenotype of HT29 cells. ROCK and MSK inhibition also abrogates the induction of 1,25(OH)2D3 24-hydroxylase (CYP24), E-cadherin, and vinculin and the repression of cyclin D1 by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 does not promote the localization of the tight junction protein occludin at the plasma membrane in cells expressing a dominant negative RhoA (N19-RhoA). In addition, 1,25(OH)2D3 specifically increases the level of the cysteine protease-inhibitor cystatin D, whereas that of cystatin SN is unaffected. The increase of cystatin D protein caused by 1,25(OH)2D3 is abrogated in N19-RhoA cells. Thus, activation of the RhoA-ROCK-p38MAPK-MSK signaling pathway is essential for the regulation of the phenotype and of the CST5/cystatin D candidate tumor suppressor and other target genes by 1,25(OH)2D3 in colon cancer cells.
The nuclear receptor superfamily mediates the regulatory activities of many hormones, nutrients and metabolites on the homeostasis and physiology of cells and tissues. Classically, ligand binding induced the ability of nuclear receptors to modulate the transcription rate of target genes (genomic effects), which led to consider them as ligand-activated transcription factors. Later, rapid actions of nuclear receptor ligands were reported that did not involve changes in gene expression. These (non-genomic) effects have been attributed in some cases to receptors different to those mediating gene transcription but most evidences indicate that they result from the activity of a population of nuclear receptor molecules acting outside the cell nucleus. Recent studies on estrogen and vitamin D, and their receptors (ERalpha/beta, VDR) support now the idea that non-genomic and genomic effects may integrate in a unique mode of action of nuclear receptor ligands, in which the non-genomic effects constitute signaling pathways required for the effects at the genome level. Here, we will discuss these novel findings and also those indicating transcriptional regulation through ligand-dependent and -independent crosstalk of nuclear receptors with beta-catenin or VDR-interacting repressor (VDIR).
The Wnt–?-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–?-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of ?-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear ?-catenin content. Nuclear ?-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–?-catenin signaling. In the presence of high nuclear ?-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the ?-catenin status of patients carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.
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