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. 2010 Dec 16;29(50):6533-42.
doi: 10.1038/onc.2010.385. Epub 2010 Aug 30.

The role of fibroblast Tiam1 in tumor cell invasion and metastasis

K Xu  1 , S Rajagopal, I Klebba, S Dong, Y Ji, J Liu, C Kuperwasser, J A Garlick, S P Naber, R J Buchsbaum
Affiliations

The role of fibroblast Tiam1 in tumor cell invasion and metastasis

K Xu et al. Oncogene. .

Abstract

The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knockout mouse models. Conversely, tumors arising in Tiam1 knockout mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with pre-malignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may have a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Effect of Tiam1 suppression in mammary fibroblasts on epithelial cell invasion in spheroid co-cultures
A. Co-cultures of HMECs and RMFs with either control (C) or suppressed (sh) levels of Tiam1 were established in Matrigel in all four possible combinations: C- HMEC in 1, 3; sh HMEC in 2,4; C-RMF in 1,2; shRMF in 3,4. Representative images were taken on day 10. Arrows indicate examples of projections extending out beyond the spheroid perimeter. B. For projection measurements, the longest projection on each spheroid from the tip of the projection to the perimeter of the spheroid was measured. Numbers along X-axis correspond to the co-culture combinations from A. Data represent mean +/- S.D. from 10 representative spheroids in each of 3 separate experiments. * indicates p-value < 0.0005, ** indicates p-value < 0.00005 by two-tailed t-Test.
Figure 2
Figure 2. Effect of Tiam1 suppression in dermal fibroblasts on keratinocyte invasion in 3-dimensional human skin equivalents (HSE)
A. HSE were established using either parental HaCat-ras-II-4 (left panels) or HaCat-ras-II-4-DN-ECad (right panels) as the epithelial keratinocyte layers, over collagen layers containing either parental fibroblasts (top panels), fibroblasts with control hairpin vector (middle panels) or shTiam1 fibroblasts (bottom panels). Arrows indicate examples of keratinocytes invading as projections from the dermal layer, asterisks indicate examples of invading cell clusters that have separated from the dermal layer. B. Numbers of invading keratinocytes present in projections or as clusters of cells separated from the epithelial layer were counted in fixed sections of HSE established over collagen layers containing either parental (WT), control vector (C), or shTiam1 (sh) fibroblasts. Data represent mean +/- S.D. for 15 representative high-power fields in at least 2 sections for each experimental condition. * indicates p-value < 0.00001 by two-tailed t-Test. Results are representative of duplicate experiments.
Figure 3
Figure 3. Effect of human stromal fibroblasts on tumor growth
Tumor size in largest dimension shown for mice implanted with SUM1315-GFP/luc only (diamonds), or co-mixed with either control RMFs (squares) or shTiam-RMFs (triangles). Each curve represents means +/- S.D. from cohorts of 10 mice.
Figure 4
Figure 4. Effect of Tiam1 suppression in stromal fibroblasts on breast cancer invasiveness
Histopathology (hematoxylin & eosin) from orthotopic tumors in mice implanted with SUM1315-GFP/luc breast cancer cells alone (top panels), co-mixed with control RMF (middle panels) or shTiam1-RMF (bottom panels). Right panels are magnification of a section from corresponding left panels depicting a representative tumor-stroma interface. T indicates primary tumor, S indicates adjacent stroma. Asterisks indicate murine mammary structures.
Figure 5
Figure 5. Immunohistochemical staining on orthotopic tumors from mice implanted and labeled as shown in Figure 4
Vimentin staining, 20x magnification. T indicates tumor cells, S indicates adjacent stroma.
Figure 6
Figure 6. Effect of Tiam1 suppression in stromal fibroblasts on breast cancer metastasis
Histopathology on lung sections from mice implanted as in Figure 4. Left panels shown at 20x magnification, thick arrows indicate nodular metastatic deposits detectable on H&E staining. Right panels show corresponding vimentin staining at 20x magnification, thin arrows indicate larger metastatic deposits, asterisks indicate examples of isolated tumor cells detectable only with vimentin staining.
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