Tetrathiomolybdate inhibits head and neck cancer metastasis by decreasing tumor cell motility, invasiveness and by promoting tumor cell anoikis

<p>Abstract</p> <p>Background</p> <p>The metastatic spread of solid tumors is directly or indirectly responsible for most cancer-related deaths. Tumor metastasis is very complex and this process requires a tumor cell to acquire enhanced motility, invasiveness and anoikis resistance to successfully establish a tumor at a distal site. Metastatic potential of tumor cells is directly correlated with the expression levels of several angiogenic cytokines. Copper is a mandatory cofactor for the function of many of these angiogenic mediators as well as other proteins that play an important role in tumor cell motility and invasiveness. We have previously shown that tetrathiomolybdate (TM) is a potent chelator of copper and it mediates its anti-tumor effects by suppressing tumor angiogenesis. However, very little is known about the effect of TM on tumor cell function and tumor metastasis. In this study, we explored the mechanisms underlying TM-mediated inhibition of tumor metastasis.</p> <p>Results</p> <p>We used two <it>in vivo </it>models to examine the effects of TM on tumor metastasis. Animals treated with TM showed a significant decrease in lung metastasis in both <it>in vivo </it>models as compared to the control group. In addition, tumor cells from the lungs of TM treated animals developed significantly smaller colonies and these colonies had significantly fewer tumor cells. TM treatment significantly decreased tumor cell motility and invasiveness by inhibiting lysyl oxidase (LOX) activity, FAK activation and MMP2 levels. Furthermore, TM treatment significantly enhanced tumor cell anoikis by activating p38 MAPK cell death pathway and by downregulating XIAP survival protein expression.</p> <p>Conclusions</p> <p>Taken together, these results suggest that TM is a potent suppressor of head and neck tumor metastasis by modulating key regulators of tumor cell motility, invasiveness and anoikis resistance.</p

A gene for the suppression of anchorage independence is located in rat chromosome 5 bands q22-23, and the rat alpha-interferon locus maps at the same region.

Cell hybrids between malignant mouse hepatoma cells and normal rat fibroblasts with approximately one set of chromosomes from each parent exhibited remarkable karyotypic stability. Most chromosomes of both parents were retained even after prolonged culture in vitro. Normally, such hybrids showed suppression of the transformed phenotype and formed no colonies in soft agar. However, two hybrids, BS140 and BS181, formed a few colonies in soft agar when many cells were seeded, and also occasional foci of cells were detected piling up in monolayer cell cultures. We isolated soft agar colonies (a-subclones) and sub-clones from foci (h-subclones) of both hybrids, and, as a control, subclones of cells from random areas without foci of one hybrid (BS181 p-subclones). When tested for soft agar growth, cells from the a- and h-subclones of both BS140 and BS181 formed colonies at frequencies comparable to the malignant mouse hepatoma parent, whereas the control cells of the BS181 p-subclones (like the normal rat parental cells) yielded no soft agar colonies. All the cell lines were subjected to detailed karyotype analysis in G-banding, which resulted in the finding that cells from the original BS140 hybrid contained at least one copy of each rat chromosome, whereas BS140 a- and h-subclones had lost both copies of rat chromosome 5. Similarly, the original BS181 hybrid contained at least one copy of each rat chromosome, whereas BS181 a- and h-subclones displayed a deletion of the segment q22-23 of rat chromosome 5. In contrast, the control BS181 p-subclones contained one or two copies of non-deleted rat chromosome 5. The conclusion is that a gene for the suppression of anchorage independence is located in the segment 5q22-23. We propose to call this gene SAI1 (for suppression of anchorage independence). Using Southern blotting, we tested whether any of several gene probes, known to correspond to DNA sequences in rat chromosome 5, were homologous to sequences in the deletion. Only one probe, corresponding to the active alpha1-interferon gene, was shown to be located within the deletion. Hence, the SAI1 gene is closely linked to the alpha 1-interferon gene, and might be identical to this locus.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

RhoC regulates cancer stem cells in head and neck squamous cell carcinoma by overexpressing IL-6 and phosphorylation of STAT3.

In this study we investigated the correlation between RhoC expression and cancer stem cells (CSCs) formation in head and neck squamous cell carcinoma (HNSCC). The inhibition of RhoC function was achieved using shRNA. The expression of stem cell surface markers, ALDH and CD44 were significantly low in two RhoC depleted HNSCC cell carcinoma cell lines. Furthermore, a striking reduction in tumorsphere formation was achieved in RhoC knockdown lines. The mRNA expression of RhoC in RhoC knockdown adherent and tumorspheres are dramatically down regulated as compared with the scrambled control. The mRNA expression of stem cell transcription factors; nanog, oct3/4 (Pouf1), and sox2 were significantly depleted in RhoC knockdown clones. Further, the phosphorylation of STAT3(ser727), and STAT3(tyr705) were significantly down regulated in RhoC knockdown clones. The overexpression of STAT3 in RhoC knockdown did not show any change in expression patterns of either-STAT3(tyr705) or stem cell transcription factors, signifying the role of RhoC in STAT3 activation and thus the expression of nanog, oct3/4 and sox2 in HNSCC. The expression of Inter leukin-6 (IL-6) in RhoC knockdown HNSCC cell lines was dramatically low as compared to the scrambled control. Further, we have shown a rescue in STAT3 phosphorylation by IL-6 stimulation in RhoC knockdown lines. This study is the first of its kind to establish the involvement of RhoC in STAT3 phosphorylation and hence in promoting the activation of core cancer stem cells (CSCs) transcription factors. These findings suggest that RhoC may be a novel target for HNSCC therapy

The rat gene map.

Journal Articleinfo:eu-repo/semantics/publishe

Somatic cell genetic analysis of growth control

info:eu-repo/semantics/publishe

A sketch diagram showing the probable steps involved in nanog activation by RhoC, mediated through the IL-6 and JAK/STAT3 pathway in head and neck cancer metastasis.

<p>A sketch diagram showing the probable steps involved in nanog activation by RhoC, mediated through the IL-6 and JAK/STAT3 pathway in head and neck cancer metastasis.</p

Expression of phospho STAT3 in scrambled control and RhoC knockdown HNSCC cell lines.

<p>(A and B) Western blot analysis showing the phosphorylation of STAT3^ser727 and STAT3 ^tyr705 in the scrambled controls and the RhoC knockdown UM-SCC-1 and -47 respectively. Normalized value with respect to total STAT3 is given as the numerical values. Remarkable decreases in the phosphorylation of STAT3 were seen in the RhoC knock-down UM-SCC-1 and -47 cell lines respectively. (C) Protein analysis shows the p-STAT3^tyr705 in the ectopically overexpressed (OE) scrambled control and the RhoC knockdown UM-SCC-1. Phosphorylated form was detected only in the scrambled control when STAT3 was over expressed. A thick band of total STAT3 can be seen in the lower panel, confirming the successful transfection of STAT3 in these clones. (D) Real time RT-PCR showing the expression of STAT3, sox2, oct3/4 and nanog before and after the STAT3 over expression in the RhoC knockdown clone. The mRNA expression of STAT3 was dramatically high after it's over expression, while no significant change in the expression was observed in sox2, oct3/4 and nanog in the RhoC knockdown clone.</p

ALDH expression and tumorsphere formation in scrambled control and RhoC knockdown UM-SCC- cell lines.

<p>FACS analysis showing the expression of (A) ALDH in the scrambled control and the RhoC knockdown clones. (B) The tumorspheres formation in the scrambled control and the RhoC knockdown UM-SCC-1 and -47cell lines. (C) The representation of tumorspheres formation efficiency in scrambled control and RhoC knockdown UM-SCC-1 obtained in 96 well plates. A significant decrease in ALDH expression was seen. Tumorsphere formation was dramatically low in RhoC knockdown UM-SCC-1 and -47 respectively. (D) A significant decrease in the RhoC mRNA expression was obtained in the RhoC knockdown adherent cell and in the tumorspheres. (E) Stem cell transcription factors, sox2, oct3/4, and nanog showing significant down regulation in their mRNA as revealed by real time RT-PCR in the scrambled control and the RhoC knockdown spheres obtained from UM-SCC-1.</p

RhoC expression in scrambled control and RhoC knockout UM-SCC-lines.

<p>(A and D) The mRNA expression of RhoC obtained by real time RT-PCR; (B and E). Western blot analysis showing RhoC expression in the scrambled control and RhoC knockdown clones (C and F). Bar graph showing the expression of active RhoC [RhoC-GTP] in scrambled control and RhoC knockdown UM-SCC-1 and-47 respectively. A significant decrease in RhoC mRNA, protein and [RhoC-GTP] levels were obtained in the RhoC knockdown cell lines.</p

Lentivirus infection and transduction in UM-SCC- cell line.

<p>Lentivirus infected cells showing the GFP expression in UM-SCC-47. (A) Histograms of the scrambled control (sr control) and (B) RhoC knockdown (RhoC kd) obtained by flowcytometry. (C) Negative control. Representation of the GFP expression in fluorescence and bright light are shown in the bottom panels. About 90% of cells were GFP positive signifying the successful transduction of shRNA using recombinant lentivirus.</p