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.

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.

Integrin alpha6Bbeta4 inhibits colon cancer cell proliferation and c-Myc activity.

BACKGROUND: Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin beta4 subunit is up-regulated in primary colon cancer. Its partner, the integrin alpha6 subunit, exists as two different mRNA splice variants, alpha6A and alpha6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these alpha6 splice variants is still lacking. METHODS: In this work, we first analyzed the expression of integrin alpha6A and alpha6B 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 alpha6A and alpha6B on the regulation of cell proliferation in a colon cancer cell line. RESULTS: Using variant-specific antibodies, we observed that alpha6A and alpha6B 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 alpha6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed alpha6B. A relative decrease of alpha6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the alpha6A/alpha6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the alpha6A/alpha6B balance in favor of alpha6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc. CONCLUSION: The findings that the alpha6Bbeta4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its alpha6Abeta4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this alpha6Bbeta4 integrin. Taken together, these findings point out the importance of integrin variant expression in colon cancer cell biology.

Differential expression of the integrins alpha6Abeta4 and alpha6Bbeta4 along the crypt-villus axis in the human small intestine.

The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.

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

As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include beta-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 the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, beta-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.

Upregulation of a functional form of the beta4 integrin subunit in colorectal cancers correlates with c-Myc expression.

The integrin beta4 subunit has been shown to be involved in various aspects of cancer progression. The aim of the present work was to evaluate the expression of beta4 in primary colon cancers and to investigate the occurrence of a previously identified intestinal nonfunctional variant of beta4 (beta4ctd-) for adhesion to laminin. Immunodetection of beta4 using a panel of antibodies and RT-PCR analyses were performed on series of paired primary colon tumors and corresponding resection margins. The beta4 subunit was found to be significantly overexpressed in cancer specimens at both the protein and transcript levels. Surprisingly, beta4 levels of expression were closely correlated with those of the oncogene c-Myc in individual specimens. In vitro studies of c-Myc overexpression showed an upregulation of beta4 promoter activity. Finally, the beta4ctd- form was identified in the normal proliferative colonic cells but was found to be predominantly absent in colon cancer cells, both in situ and in vitro. We concluded that the beta4ctd- form is lost from colon cancer cells, while the level of the wild-type form of beta4, which is functional for adhesion to laminin, is increased in primary tumors in relation with the expression of c-Myc.

Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms.

We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominant-negative mutants) or beta1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants. Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on beta1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on beta1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation state-specific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.

Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis.

BACKGROUND & AIMS: Little is known of the signaling events implicated in the induction of human enterocytic anoikis. In the present study, we analyzed the role of the stress-activated protein kinase p38 in this process. METHODS: Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of a dominant negative form) or beta1 integrins (antibody blocking), or by maintaining cells in suspension. Expression/activation parameters of p38 (isoforms alpha, beta, gamma, delta) and of the Fak/phosphatidylinositol-3-kinase (PI3-K)/Akt anoikis-suppressing pathways were analyzed. Kinase activities of p38 isoforms also were blocked by pharmacologic inhibitors or by overexpression of dominant-negative forms. RESULTS: (1) p38 activation is sustained transiently after induction of anoikis in both undifferentiated and differentiated enterocytes; (2) such sustenance of p38 activation is associated with a down-regulation of the Fak/PI3-K/Akt pathway; (3) distinct profiles of p38 isoform expression are exhibited by undifferentiated (alpha, beta, gamma) and differentiated (alpha, gamma, delta) enterocytes; (4) none of the 4 known p38 isoforms was found to promote cell survival in either differentiation state; and (5) only p38beta and p38delta are required specifically for anoikis in undifferentiated and differentiated cells, respectively. CONCLUSIONS: Distinct p38 isoforms play a major role in the induction of enterocytic anoikis and the regulation of such selective p38 isoform-mediated anoikis is linked with the state of cell differentiation. These data provide novel insights into the synchronized regulation of cell survival/death required in the epithelial renewal process along the human intestinal crypt-villus axis.