Diversity of Matriptase Expression Level and Function in Breast Cancer

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent

Increases in c-Yes Expression Level and Activity Promote Motility But Not Proliferation of Human Colorectal Carcinoma Cells12

Increases in the levels and/or activity of nonreceptor tyrosine kinases c-Src and c-Yes are often associated with colorectal carcinogenesis. The physiological consequences of increased c-Yes activity during the early and late stages of tumorigenesis, in addition to the degree of redundancy between c-Yes and c-Src in colorectal cancer cells, remain elusive. To study the consequences of increases in c-Yes levels and activity in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Yes levels and activity can be inducibly increased by a tightly controlled expression of wild-type c-Yes or by constitutively active mutants of c-Yes, c-YesY537F, and c-YesΔt6aa. c-Yes induction resulted in increased cell motility but did not promote proliferation either in vitro or in vivo. These results suggest that in later stages of colorectal carcinogenesis, elevations in c-Yes levels/activity may promote cancer spread and metastasis rather than tumor growth

Comparison of Doxycycline Delivery Methods for Tet-Inducible Gene Expression in a Subcutaneous Xenograft Model

Doxycycline (Dox) controlled Tet systems provide a powerful and commonly used method for functional studies on the consequences of gene overexpression/downregulation. However, whereas Dox delivery in tissue culture in vitro is relatively simple, the situation in vivo is more complex. Several methods of Dox delivery in vivo have been described—e.g., in drinking water containing alcohol, in drinking water containing various concentrations of sucrose, and in feed. Unfortunately there are no reports directly comparing the advantages and disadvantages of these diverse methods, and there is no generally accepted standard. We therefore compared four non-invasive methods of Dox delivery in vivo—in drinking water, by gavage, as a jelly, and in standard feed. To assess the delivery of Dox by these methods, we used a subcutaneous xenograft model based on colorectal carcinoma cells engineered for Dox-inducible expression of an activated mutant of c-Src and the luciferase reporter gene. Our results indicate that feed represents the most favorable method of Dox administration

Protein kinase C delta is phosphorylated on five novel Ser/Thr sites following inducible overexpression in human colorectal cancer cells

Phosphorylation plays an important role in regulation of protein kinase C delta (PKCδ). To date, three Ser/Thr residues (Thr 505, Ser 643, and Ser 662) and nine tyrosine residues (Tyr 52, Tyr 64, Tyr 155, Tyr 187, Tyr 311, Tyr 332, Tyr 512, Tyr 523, and Tyr 565) have been defined as regulatory phosphorylation sites for this protein (rat PKCδ numbering). We combined doxycycline-regulated inducible gene expression technology with a hypothesis-driven mass spectrometry approach to study PKCδ phosphorylation pattern in colorectal cancer cells. We report identification of five novel Ser/Thr phosphorylation sites: Thr 50, Thr 141, Ser 304, Thr 451, and Ser 506 (human PKCδ numbering) following overexpression of PKCδ in HCT116 human colon carcinoma cells grown in standard tissue culture conditions. Identification of potential novel phosphorylation sites will affect further functional studies of this protein, and may introduce additional complexity to PKCδ signaling

Blocking phosphoinositide 3-kinase activity in colorectal cancer cells reduces proliferation but does not increase apoptosis alone or in combination with cytotoxic drugs

In response to growth factors, class IA phosphoinositide 3-kinases (PI3Ks) phosphorylate PtdIns(4,5)P(2), converting it to PtdIns(3,4,5)P(3) to activate protein kinase B/Akt. This is widely reported to promote tumorigenesis via increased cell survival, proliferation, migration and invasion and many tumor types, including colorectal cancer, exhibit increased PI3K signaling. In order to investigate the effect of inhibiting PI3K and as an alternative to the use of small molecular inhibitors of PI3K with varying degrees of selectivity, HT29 and HCT116 colorectal cancer cells bearing mutant PIK3CA were generated that could be induced with doxycycline to express synchronously a dominant negative subunit of PI3K, Δp85α. Upon induction, decreased levels of phosphorylated PKB were detected, confirming PI3K signaling impairment. Induction of Δp85α in vitro reduced cell number via accumulation in G(0)/G(1) phase of the cell-cycle in the absence of increased apoptosis. These effects were recapitulated in vivo; HT29 cells expressing Δp85α and grown as tumor xenografts had a significantly slower growth rate upon administration of doxycycline with reduced Ki67 staining without increased levels of apoptotic tissue biomarkers. Furthermore, in vitro Δp85α expression did not sensitize HT29 cells to oxaliplatin- or etoposide-induced apoptosis, irrespective of drug treatment schedule. Further analysis comparing isogenic HCT116 cells with and without mutation in PIK3CA showed no impact of the mutation in either proliferative or apoptotic response to PI-3K inhibition. These data demonstrate in colorectal cancer cells that PI3K inhibition does not provoke apoptosis per se nor enhance oxaliplatin- or etoposide-induced cell death

Increases in c-Src Expression Level and Activity Do Not Promote the Growth of Human Colorectal Carcinoma Cells In Vitro and In Vivo

The levels and activity of c-Src in colorectal cancer cells increase steadily during the course of colorectal carcino-genesis and are most highly elevated in advanced metastatic disease. However, the effects of increases in c-Src activity on the proliferation of colorectal cancer cells during early and late stages of tumorigenesis remain elusive. To study the consequences of increases in c-Src levels and activity on the growth of colorectal cancer cells in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Src levels and activity could be inducibly increased by a tightly controlled expression of wild-type c-Src or of the constitutively active mutant of c-Src, c-SrcY527F. Src induction activated multiple signaling pathways (often associated with a proliferative response) but promoted neither cell proliferation in vitro nor tumor growth in a xenograft model in vivo. These results indicate that, in more advanced stages of colorectal carcinogenesis, increases in c-Src levels and activity are likely to have functions other than the direct promotion of tumor growth