The Tongue Squamous Carcinoma Cell Line Cal27 Primarily Employs Integrin α6β4-Containing Type II Hemidesmosomes for Adhesion Which Contribute to Anticancer Drug Sensitivity

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVβ3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVβ3 expressing Cal27-derived cell clone 2B1, αVβ5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6β4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6β4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6β4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVβ3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6β4 not occurred. Taken together, our results identify a key role for α6β4-containing type II hemidesmosomes in regulating anticancer drug sensitivity

Development of a low-seroprevalence, αvβ6 integrin-selective virotherapy based on human adenovirus type 10

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre- existing immunity will be beneficial for future clinical translation. We generated a low- seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin-selective peptide, A20, to target αvβ6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines demonstrated significantly increased transduction mediated by αvβ6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvβ6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvβ6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation

Distribution of Income and Taxes in Slovenia and Croatia

The purpose of this article is to estimate the redistributive effects of personal income tax (PIT) in Slovenia and Croatia. The decomposition of the Gini coefficient developed by Aronson, Johnson & Lambert reveals only a limited difference between potential and actual redistribution, with this loss being a consequence of the different tax treatment of taxpayers with equal pre-tax income. The results also suggest that Croatia experiences greater income inequality among taxpayers than Slovenia. Another decomposition of inequality measure indicates that some types of income — especially wages — contribute a constant and high proportion to the overall inequality seen in both countries during the period examined.

Working for 200 Euro? The Unintended Effects of Traineeship Reform on Youth Labor Market Outcomes

This paper evaluates the effects of an active labor market policy (ALMP) reform, the so-called SOR measure (vocational training for work without commencing employment), on youth labor market outcomes in the newest EU member state—Croatia. In 2012, SOR was redesigned to ease the first labor market entry and promote on-the-job training, enabling a young person without relevant work experience to get a one-year contract and a net monthly remuneration of 210 euro, while after 2014, the measure also became a part of the European Youth Guarantee. Pooling Croatian Labor Force Surveys from 2007 to 2016 and using the difference-in-difference strategy, we estimate the causal intent-to-treat effect of the program reform on labor market outcomes. The main results indicate that the reform has had, at best, neutral effects on employment and unemployment, while there is evidence that a portion of young individuals was propelled into inactivity. Though expected, adverse effect on wages—both at the mean and at higher percentiles of the wage distribution—is driven mostly by wages received by women and university graduates

KANK2 links αVβ5 focal adhesions to microtubules and regulates sensitivity to microtubule poisons and cell migration

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin alpha V specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined alpha V-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin alpha V specific shRNA-expressing cell clones with decreased expression of integrin alpha V. MS analysis showed that cells preferentially use integrin alpha V beta 5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin alpha V identified key components of integrin alpha V beta 5 adhesion complexes as talins 1 and 2, alpha-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins alpha and beta, ELKS, LL5b, MACF1, KANK1, and KANK2), following alpha V knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin alpha V knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin alpha V beta 5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications