Integrin α6Bβ4 inhibits colon cancer cell proliferation and c-Myc activity

<p>Abstract</p> <p>Background</p> <p>Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin β4 subunit is up-regulated in primary colon cancer. Its partner, the integrin α6 subunit, exists as two different mRNA splice variants, α6A and α6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these α6 splice variants is still lacking.</p> <p>Methods</p> <p>In this work, we first analyzed the expression of integrin α6A and α6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of α6A and α6B on the regulation of cell proliferation in a colon cancer cell line.</p> <p>Results</p> <p>Using variant-specific antibodies, we observed that α6A and α6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express α6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed α6B. A relative decrease of α6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the α6A/α6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the α6A/α6B balance in favor of α6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc.</p> <p>Conclusion</p> <p>The findings that the α6Bβ4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its α6Aβ4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this α6Bβ4 integrin. Taken together, these findings point out the importance of integrin variant expression in colon cancer cell biology.</p

Integrin [alpha]6A[béta]4 in colon cancer

Les niveaux d'expression de l'intégrine a.6P4 sont reconnus pour être élevés dans un certain nombre de cancers humains. Dans ce contexte pathologique, cette intégrine exerce un rôle stimulateur à plusieurs niveaux de la progression tumorale, tels la promotion de l'invasion et l'activation de voies de signalisation intracellulaires associées à la prolifération et la survie cellulaire. Dans ce travail, les niveaux d'expression de la sous-unité intégrine P4 dans des adénocarcinomes de colon humain ont été évalués par quantification de l' ARN messager, en comparaison avec les niveaux d'expression de P4 dans les régions saines du colon de chaque patient. Non seulement le messager encodant f34 se trouve surexprimé dans les tissus cancéreux, mais les niveaux d'expression de P4 corrèlent aussi avec l'expression du facteur de transcription oncogénique MYC. Cette corrélation suggère une importance fonctionnelle pour les niveaux d'expression de la sous-unité P4 compte tenu que la surexpression de MYC dans une lignée cellulaire de cancer du colon stimule l'activité transcriptionnelle d'une variété de constructions du promoteur de p4. Le pré-ARNm de la sous unité intégrine a.6 subit un épissage alternatif conduisant à deux variants différents, soit a6A et a6B. Ceux-ci diffèrent par leurs domaines intracellulaires et leurs propriétés biochimiques. Afin de caractériser le profil d'expression de l'intégrine a6p4 dans les cancers du colon, les niveaux totaux d'expression de a.6 ainsi que ceux des deux variants d'épissage alternatif furent étudiés. Tout comme dans le cas de la sous-unité p4, les niveaux totaux de a.6 sont augmentés dans les cas de cancers comparativement aux marges de résection correspondantes. De manière intéressante, une altération dans les ratios d'expression des deux variants de a.6 a été remarquée dans 81 % des cancers étudiés. Ainsi, ces ratios passent d'une expression prédominante de a6B dans les marges de résection saines, à une expression prédominante de a6A dans les adénocarcinomes primaires. Une telle expression différentielle des deux variants d'épissage alternatif est aussi retrouvée dans l'intestin grêle humain normal. En effet, les cellules cryptales prolifératives expriment le variant a6A, tandis que le compartiment différencié de l'axe crypte-villosité exprime plutôt le variant a6B. L'importance fonctionnelle de ces observations est confirmée par la surexpression des deux variants de a.6, conduisant à une activation différentielle des complexes transcriptionnels P-caténine!fCF4 et MYC, lesquels sont des joueurs importants dans la prolifération des cellules intestinales et la formation des adénocarcinomes

Integrin [alpha]6A[béta]4 in colon cancer

The expression level of the [alpha]6A[béta]4 integrin has been reported to be elevated in a number of human cancers in accordance with its known stimulatory role in several aspects of cancer progression such as promoting invasion and stimulating intracellular signaling of importance for cellular proliferation and survival. In the present work the expression levels of [béta]4 in adenocarcinomas of the human colon were evaluated at the transcript level in comparison with the expression levels in patient matched healthy colonic tissue. Not only were the expression level of the [béta]4 subunit found to be upregulated in cancerous tissue, but levels of expression of [béta]4 were found to be closely correlated to levels of expression of those of the oncogenic transcription factor MYC. This correlation was demonstrated to be of functional importance for the expression level of [béta]4, since forced expression of MYC in a colon cancer cell line stimulated transcriptional activity of various [béta]4 promoter constructs.The pre-mRNA of the [alpha]6 subunit undergo alternative splicing yielding two different variants: the A and the B variants. Their distinct intracellular domains and their distinct biochemical properties characterize these variants. As part of the characterization of the expression of the [alpha]6A[béta]4 integrin in colon cancers, the expression level of total [alpha]6 as well as the two different variants were investigated. As for the [béta]4 subunit the total level of [alpha]6 were found to be elevated in cancers as compared to their corresponding healthy resection margins. Interestingly, an alteration of the ratio of expression of two splice variants of the [alpha]6 subunit was found to be significantly altered in 81% of the cancers investigated. Thus, the expression ratio shifted from a predominant expression of [alpha]6B in the healthy resection margins to a predominant expression of [alpha]6A in the primary adenocarcinomas. Such differential expression of splice variants was also found in the normal human small intestine. Indeed, the proliferative crypts were found to express the [alpha]6A variant, while the differentiated compartments of the crypt-villus axis were found to express the [alpha]6B variant.The functional importance of this was confirmed when over-expression of the two different splice variants were demonstrated to differentially activate the [béta]-catenin/TCF4 and MYC transcriptional complexes, two well known players in intestinal proliferation and adenocarcinomas formation. On the other hand the two splice variants were not found to differentially affect differentiation of human enterocytes as assayed by monitoring the transcriptional activity from promoter constructs of sucrase-isomaltase, lactasephlorizin hydrolase, DPPIV and ALPP. Finally, it has become increasingly clear that the classic normalizing genes such as GAPD and [béta]-actin can display modulated expression levels across tissue types and during cellular differentiation. Therefore, experiments aimed at identifying and validating normalizing genes for transcript measurements during the complex cellular processes of differentiation and cancer progression in the human gut were carried out. Previously generated microarray data were screened for candidate genes of which two were selected for further analyses along with seven commonly used normalizing genes. Real time RT-PCR amplifying the nine putative normalizing genes were performed on various samples representing different differentiation stages of human enterocytes as well as on patient matched primary tumors and healthy resection margins. Using several statistical approaches, RPLP0 and B2M were found to be the two best normalizing genes for experiments aimed at assessing transcript during differentiation and in cancers respectively. These studies should serve as a paradigm for other studies aimed at validating other normalizing genes under other experimental settings

Innovative Methodology Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon

quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon. Am J Physiol Gastrointest Liver Physiol 290: G1067–G1074, 2006. First published January 6, 2006; doi:10.1152/ajpgi.00234.2005.—As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include �-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined th

Clinical outcomes of ALK+ non-small cell lung cancer in Denmark

BACKGROUND: Real-world clinical outcomes of anaplastic lymphoma kinase positive (ALK+) non-small cell lung cancer (NSCLC) patients vary. This study aimed to investigate the treatment and clinical outcomes of all ALK+ NSCLC patients in Denmark in the period 2011-2018, regardless of disease stage.MATERIALS AND METHODS: A national pathology database with complete coverage was used to identify ALK+ NSCLC patients diagnosed between 2011 and 2018. Clinical data were obtained through retrospective chart reviews. Overall survival (OS) and duration of treatment (DOT) were analyzed using Kaplan-Meier methodologies.RESULTS: A total of 209 ALK+ NSCLC patients were included. The cohort had a slight overrepresentation of female patients (56.5%) with a mean age of 61.6 years. Most patients were adenocarcinoma cases (97%) and presented with an ECOG performance status of 0-1 (79%). Stage IIIb-IVb patients comprised 70% of the cohort. The use of ALK-tyrosine kinase inhibitors (TKIs) as first-line treatment increased over time, with the 1st generation ALK-TKI crizotinib being the predominant treatment in the 1st line. In 1st line treatment, 2nd generation ALK-TKIs had a median DOT more than twice the median DOT of crizotinib (25.1 and 9.1 months, respectively). The median OS for the entire cohort was 44.0 months. Patients with stage I-IIIA disease had a median OS that had not been reached, while those with stage IIIb-IVb disease had a median OS of 31.8 months. Patients with stage IIIb-IVb disease receiving an ALK-TKI as 1st line treatment had a median OS of 42.5 months with immature follow-up. Brain metastases at diagnosis or choice of 1st line treatment did not statistically significantly impact OS.CONCLUSION: This study gives insights into the treatment and outcome of ALK+ NSCLC patients in Denmark and provides a real-world confirmation of the superior disease control provided by 2nd generation ALK-TKIs as compared to the 1st generation ALK-TKI crizotinib.</p

Clinical outcomes of <i>ALK</i>+ non-small cell lung cancer in Denmark

Real-world clinical outcomes of anaplastic lymphoma kinase positive (ALK+) non-small cell lung cancer (NSCLC) patients vary. This study aimed to investigate the treatment and clinical outcomes of all ALK+ NSCLC patients in Denmark in the period 2011–2018, regardless of disease stage. A national pathology database with complete coverage was used to identify ALK+ NSCLC patients diagnosed between 2011 and 2018. Clinical data were obtained through retrospective chart reviews. Overall survival (OS) and duration of treatment (DOT) were analyzed using Kaplan-Meier methodologies. A total of 209 ALK+ NSCLC patients were included. The cohort had a slight overrepresentation of female patients (56.5%) with a mean age of 61.6 years. Most patients were adenocarcinoma cases (97%) and presented with an ECOG performance status of 0–1 (79%). Stage IIIb–IVb patients comprised 70% of the cohort. The use of ALK-tyrosine kinase inhibitors (TKIs) as first-line treatment increased over time, with the 1st generation ALK-TKI crizotinib being the predominant treatment in the 1st line. In 1st line treatment, 2nd generation ALK-TKIs had a median DOT more than twice the median DOT of crizotinib (25.1 and 9.1 months, respectively). The median OS for the entire cohort was 44.0 months. Patients with stage I–IIIA disease had a median OS that had not been reached, while those with stage IIIb–IVb disease had a median OS of 31.8 months. Patients with stage IIIb–IVb disease receiving an ALK-TKI as 1st line treatment had a median OS of 42.5 months with immature follow-up. Brain metastases at diagnosis or choice of 1st line treatment did not statistically significantly impact OS. This study gives insights into the treatment and outcome of ALK+ NSCLC patients in Denmark and provides a real-world confirmation of the superior disease control provided by 2nd generation ALK-TKIs as compared to the 1st generation ALK-TKI crizotinib.</p

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α–mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation