SDA and IDA - Two aptamers to inhibit cancer cell adhesion.

Aptamers which bind to proteins involved in cell-cell interactions could have significant value to directly affect cancer cell adhesion or for directed cargo delivery. Here, I discuss two aptamers: aptamer SDA which binds to E- and P-selectin, and aptamer IDA which binds to α6ß4 integrin. Both aptamers (SDA 91 nt and IDA 77 nt) bind their target proteins with dissociation constants in the 100-150 nM range and substantially inhibit special cellular adhesion, possibly a first and pivotal step in transendothelial migration during metastasis formation. The aptamers' half-lives in cell culture media are between two and six hours. IDA is internalized by integrin presenting cells within minutes thus possibly serving as vehicle for directed cargo delivery.

Classification of 27 Tumor-Associated Antigens by Histochemical Analysis of 36 Freshly Resected Lung Cancer Tissues.

In previous studies, we identified 29 tumor-associated antigens (TAAs) and isolated 488 human monoclonal antibodies (mAbs) that specifically bind to one of the 29 TAAs. In the present study, we performed histochemical analysis of 36 freshly resected lung cancer tissues by using 60 mAbs against 27 TAAs. Comparison of the staining patterns of tumor cells, bronchial epithelial cells, and normal pulmonary alveolus cells and interalveolar septum allowed us to determine the type and location of cells that express target molecules, as well as the degree of expression. The patterns were classified into 7 categories. While multiple Abs were used against certain TAAs, the differences observed among them should be derived from differences in the binding activity and/or the epitope. Thus, such data indicate the versatility of respective clones as anti-cancer drugs. Although the information obtained was limited to the lung and bronchial tube, bronchial epithelial cells represent normal growing cells, and therefore, the data are informative. The results indicate that 9 of the 27 TAAs are suitable targets for therapeutic Abs. These 9 Ags include EGFR, HER2, TfR, and integrin α6ß4. Based on our findings, a pharmaceutical company has started to develop anti-cancer drugs by using Abs to TfR and integrin α6ß4. HGFR, PTP-LAR, CD147, CDCP1, and integrin αvß3 are also appropriate targets for therapeutic purposes.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Suppression of anoikis in human intestinal epithelial cells: differentiation state-selective roles of α2ß1, α3ß1, α5ß1, and α6ß4 integrins.

BACKGROUND: Regulation of anoikis in human intestinal epithelial cells (IECs) implicates differentiation state-specific mechanisms. Human IECs express distinct repertoires of integrins according to their state of differentiation. Therefore, we investigated whether α2ß1, α3ß1, α5ß1, and α6ß4 integrins perform differentiation state-specific roles in the suppression of IEC anoikis. RESULTS: Human (HIEC, Caco-2/15) IECs were exposed to specific antibodies that block the binding activity of integrin subunits (α2, α3, α5, α6, ß1 or ß4) to verify whether or not their inhibition induced anoikis. The knockdown of α6 was also performed by shRNA. Additionally, apoptosis/anoikis was induced by pharmacological inhibition of Fak (PF573228) or Src (PP2). Anoikis/apoptosis was assayed by DNA laddering, ISEL, and/or caspase activity (CASP-8, -9, or -3). Activation levels of Fak and Src, as well as functional Fak-Src interactions, were also assessed. We report herein that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones. This involves an earlier onset of anoikis when kept in suspension, as well as significantly greater contributions from ß1 and ß4 integrins in the suppression of anoikis in differentiated cells, and functional distinctions between ß1 and ß4 integrins in engaging both Fak and Src, or Src only, respectively. Likewise, Fak performs significantly greater contributions in the suppression of anoikis in differentiated cells. Additionally, we show that α2ß1 and α5ß1 suppress anoikis in undifferentiated cells, whereas α3ß1 does so in differentiated ones. Furthermore, we provide evidence that α6ß4 contributes to the suppression of anoikis in a primarily α6 subunit-dependent manner in undifferentiated cells, whereas this same integrin in differentiated cells performs significantly greater contributions in anoikis suppression than its undifferentiated state-counterpart, in addition to doing so through a dependence on both of its subunits. CONCLUSIONS: Our findings indicate that the suppression of human IEC anoikis implicates differentiation state-selective repertoires of integrins, which in turn results into distinctions in anoikis regulation, and sensitivity, between undifferentiated and differentiated IECs. These data further the functional understanding of the concept that the suppression of anoikis is subjected to cell differentiation state-selective mechanisms.

Curcumin inhibition of the functional interaction between integrin α6ß4 and the epidermal growth factor receptor.

The functional interaction between integrin α6ß4 and growth factor receptors has been implicated in key signaling pathways important for cancer cell function. However, few attempts have been made to selectively target this interaction for therapeutic intervention. Previous studies showed that curcumin, a yellow pigment isolated from turmeric, inhibits integrin α6ß4 signaling important for breast carcinoma cell motility and invasion, but the mechanism is not currently known. To address this issue, we tested the hypothesis that curcumin inhibits the functional interaction between α6ß4 and the epidermal growth factor receptor (EGFR). In this study, we found that curcumin disrupts functional and physical interactions between α6ß4 and EGFR, and blocks α6ß4/EGFR-dependent functions of carcinoma cells expressing the signaling competent form of α6ß4. We further showed that curcumin inhibits EGF-dependent mobilization of α6ß4 from hemidesmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby prevents α6ß4 distribution to lipid rafts where functional interactions between α6ß4 and EGFR occur. These data suggest a novel paradigm in which curcumin inhibits α6ß4 signaling and functions by altering intracellular localization of α6ß4, thus preventing its association with signaling receptors such as EGFR.

Mnk mediates integrin α6ß4-dependent eIF4E phosphorylation and translation of VEGF mRNA.

It was previously shown that integrin α6ß4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6ß4 regulates the activity of eIF4E through the Ser/Thr kinase Mnk. Although a role for Mnk in various aspects of cancer progression has been established, a link between integrin and Mnk activity has not. Here we show that Mnk1 is a downstream effector of integrin α6ß4 and mediates the α6ß4 signaling, important for translational control. Integrin α6ß4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6ß4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6ß4 level is high.

Curcumin inhibition of integrin (alpha6beta4)-dependent breast cancer cell motility and invasion.

Curcumin, a polyphenol natural product isolated from the rhizome of the plant Curcuma longa, has emerged as a promising anticancer therapeutic agent. However, the mechanism by which curcumin inhibits cancer cell functions such as cell growth, survival, and cell motility is largely unknown. We explored whether curcumin affects the function of integrin alpha(6)beta(4), a laminin adhesion receptor with an established role in invasion and migration of cancer cells. Here we show that curcumin significantly reduced alpha(6)beta(4)-dependent breast cancer cell motility and invasion in a concentration-dependent manner without affecting apoptosis in MDA-MB-435/beta4 (beta(4)-integrin transfectants) and MDA-MB-231 breast cancer cell lines. Further, curcumin selectively reduced the basal phosphorylation of beta(4) integrin (Y1494), which has been reported to be essential in mediating alpha(6)beta(4)-dependent phosphatidylinositol 3-kinase activation and cell motility. Consistent with this finding, curcumin also blocked alpha(6)beta(4)-dependent Akt activation and expression of the cell motility-promoting factor ENPP2 in MDA-MB-435/beta4 cell line. A multimodality approach using curcumin in combination with other pharmacologic inhibitors of alpha(6)beta(4) signaling pathways showed an additive effect to block breast cancer cell motility and invasion. Taken together, these findings show that curcumin inhibits breast cancer cell motility and invasion by directly inhibiting the function of alpha(6)beta(4) integrin, and suggest that curcumin can serve as an effective therapeutic agent in tumors that overexpress alpha(6)beta(4).

Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding.

We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 31, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3beta. We also present evidence that PV signaling is activated via the alpha6beta4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection.

Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells.

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the alpha6beta4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the alpha6beta4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced alpha6beta4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of beta4 expression in these cells augmented the formation of alpha6beta1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the alpha6beta4 integrin in invasion and migration that has been demonstrated previously by expression of the beta4 subunit in beta4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the alpha6beta4 integrin may be a useful approach to prevent carcinoma cell progression.