Pro-invasive Signaling Research Articles

Pressured to Metastasize

Interstitial fluid pressure induces autocrine proliferative and proinvasive glutamate signaling in some cancer cells.

Abstract 2342: RSK2 phosphorylates and activates CREB to promote HNSCC cell invasion through upregulation of pro-metastatic MCAM and Fascin-1

Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer and clinically effective treatment of HNSCC remains difficult due to metastasis. Therefore, it is of immerse clinical interest to identify metastasis-promoting genes in HNSCC tumors to improve prognosis and define targets for therapy. We recently reported that continued expression of p90 Ribosomal S6 kinase 2 (RSK2) contributes to the maintenance of the invasive and metastatic potential of HNSCC cells in vitro and in vivo, respectively. By a phospho-proteomics based study using a phospho-antibody microarray, we identified a spectrum of pro-metastatic proteins whose phosphorylation levels are regulated by RSK2 in HNSCC cells, including CREB, Hsp27, c-Jun, Elk-1, FAK, IRS-1, Jun-B, c-MET, and Stathmin. CREB is a transcription factor and RSK2 activates CREB by phosphorylating CREB Ser133. To explore the molecular mechanism underlying the RSK2→CREB pathway-mediated pro-invasive signals, we surveyed potential links between RSK2 and known CREB transcription targets that are implied in cell invasion and tumor metastasis, including HPSE-1, IRS-2, MMP2, VEGF, CNN/Cyr61, MCAM, and Fascin-1. RT-PCR results showed that stable knockdown of RSK2 in highly invasive HNSCC cells results in decreased mRNA levels of VEGF, MCAM and Fascin-1. MCAM (melanoma cell adhesion molecule) is one of the immunoglobulin superfamily which is known to be involved in focal adhesion assembly and cell migration. Fascin-1 is a critical hallmark of the invasive phenotype in cancer cells which bundles F-actin and contributes to cell migration and invasion. Using real-time RT-PCR, we confirmed that stable knockdown of RSK2 result in significant decrease in mRNA levels of MCAM and Fascin-1 in the RSK2-expressing, highly invasive HNSCC M4e, 212LN and 37B cells. Moreover, targeting MCAM and Fascin-1 by shRNA in 212LN cells led to significantly increased cell sensitivity to detachment-induced anoikis, which correlated with attenuated cell invasive potential of these cells. In addition, overexpression of MCAM and Fascin-1 in 212LN cells with stable knockdown of RSK2 rescued phenotypes due to lack of RSK2, resulting in increased cell invasion and reduced cell sensitivity to anoikis, compared to RSK2 knockdown cells. These data together suggest a transcription-dependent mechanism in which RSK2 may signal through CREB to provide anti-anoikis signals and promote HNSCC cell invasion, in part by up-regulated pro-metastatic proteins such as MCAM and Fascin-1. Our studies may shed new insights into understanding of molecular mechanisms underlying HNSCC metastasis and development of novel therapeutic strategies to treat metastatic HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2342. doi:10.1158/1538-7445.AM2011-2342

Abstract P4-06-16: TWEAK/Fn14 Signaling Axis as a Driver of Breast Cancer Cell Invasion and Metastasis

Abstract The molecular events associated with the multi-step process of cell invasion and metastasis are poorly understood. Tumor necrosis factor receptor family member fibroblast growth factor-inducible 14 (Fn14) and its ligand TWEAK (tumor necrosis factor-like weak inducer of apoptosis) have recently emerged as a pro-invasive signaling mechanism and indicator of poor prognosis in breast cancer. Our laboratory has recently reported enhanced Fn14 expression in a significant proportion of breast tumor samples, but not in normal breast epithelium. Analysis of publicly available breast cancer data indicates that high Fn14 expression positively correlates with HER2/neu positivity, estrogen receptor negativity, high tumor grade, positive lymph nodes, and metastasis. Modulation of Fn14 expression in breast cancer cell lines either by exogenous overexpression or siRNA-mediated blockade is sufficient to regulate breast cancer cell invasive behavior in vitro. However, the mechanisms leading to Fn14 overexpression, and how activation of the TWEAK/Fn14 signaling axis contributes to breast tumor malignant progression remain unclear. We hypothesized that genomic alterations and inappropriate autocrine or paracrine growth factor signaling may play a role in aberrant Fn14 regulation. Using array genomic hybridization and fluorescence in situ hybridization, we found increased Fn14 gene copy number relative to centromere 16 in two out of four Fn14-expressing, invasive breast cancer cell lines and in 28% of primary breast tumors. We also investigated the role of exogenous growth factors known to play important roles in the breast tumor microenvironment in stimulating Fn14 expression in vitro. TWEAK, EGF, IGF1, HRG and HGF/SF were sufficient to stimulate Fn14 expression de novo in a variety of biologic contexts. Many of these growth factors converge on the MAPK signaling axis. We have confirmed a functional role for the MAPK pathway in facilitating Fn14-mediated invasion. Consistent with this observation, Fn14 expression in primary breast tumors is high in the context of K-RAS mutation. We are currently evaluating the presence of important key regulatory regions within the Fn14 promoter that may be contributing Fn14 over expression in breast cancer cells. To define mechanisms by which Fn14 contributes to a malignant cell phenotype we have identified an Fn14-specific invasion signature using gene expression microarray profiling. Analysis of this data points to key malignant mechanisms likely to be impacted by Fn14 activation within different biologic contexts that warrants further investigation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-16.

Implication of Metastasis Suppressor NM23-H1 in Maintaining Adherens Junctions and Limiting the Invasive Potential of Human Cancer Cells

Loss of NM23-H1 expression correlates with the degree of metastasis and with unfavorable clinical prognosis in several types of human carcinoma. However, the mechanistic basis for the metastasis suppressor function of NM23-H1 is obscure. We silenced NM23-H1 expression in human hepatoma and colon carcinoma cells and methodologically investigated effects on cell-cell adhesion, migration, invasion, and signaling linked to cancer progression. NM23-H1 silencing disrupted cell-cell adhesion mediated by E-cadherin, resulting in β-catenin nuclear translocation and T-cell factor/lymphoid-enhancing factor-1 transactivation. Further, NM23-H1 silencing promoted cellular scattering, motility, and extracellular matrix invasion by promoting invadopodia formation and upregulating several matrix metalloproteinases (MMP), including membrane type 1 MMP. In contrast, silencing the related NM23-H2 gene was ineffective at promoting invasion. NM23-H1 silencing activated proinvasive signaling pathways involving Rac1, mitogen-activated protein kinases, phosphatidylinositol 3-kinase (PI3K)/Akt, and src kinase. Conversely, NM23-H1 was dispensable for cancer cell proliferation in vitro and liver regeneration in NM23-M1 null mice, instead inducing cellular resistance to chemotherapeutic drugs in vitro. Analysis of NM23-H1 expression in clinical specimens revealed high expression in premalignant lesions (liver cirrhosis and colon adenoma) and the central body of primary liver or colon tumors, but downregulation at the invasive front of tumors. Our findings reveal that NM23-H1 is critical for control of cell-cell adhesion and cell migration at early stages of the invasive program in epithelial cancers, orchestrating a barrier against conversion of in situ carcinoma into invasive malignancy.

The secretory small GTPase Rab27B as a marker for breast cancer progression.

In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.

The secretory small GTPase Rab27B as a marker for breast cancer progression.

In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.

Reduced Expression of the Hyaluronan and Proteoglycan Link Proteins in Malignant Gliomas

Malignant gliomas have a distinctive ability to infiltrate the brain parenchyma and disrupt the neural extracellular matrix that inhibits motility of axons and normal neural cells. Chondroitin sulfate proteoglycans (CSPGs) are among the major inhibitory components in the neural matrix, but surprisingly, some are up-regulated in gliomas and act as pro-invasive signals. In the normal brain, CSPGs are thought to associate with hyaluronic acid and glycoproteins such as the tenascins and link proteins to form the matrix scaffold. Here, we examined for the first time the expression of link proteins in human brain and malignant gliomas. Our results indicate that HAPLN4 and HAPLN2 are the predominant members of this family in the adult human brain but are strongly reduced in the tumor parenchyma. To test if their absence was related to a pro-invasive gain of function of CSPGs, we expressed HAPLN4 in glioma cells in combination with the CSPG brevican. Surprisingly, HAPLN4 increased glioma cell adhesion and migration and even potentiated the motogenic effect of brevican. Further characterization revealed that HAPLN4 expressed in glioma cells was largely soluble and did not reproduce the strong, hyaluronan-independent association of the native protein to brain subcellular membranes. Taken together, our results suggest that the tumor parenchyma is rich in CSPGs that are not associated to HAPLNs and could instead interact with other extracellular matrix proteins produced by glioma cells. This dissociation may contribute to changes in the matrix scaffold caused by invasive glioma cells.

Stroma reactions and cancer invasion

Stroma reactions and cancer invasion

Differential impact of TGF-β and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-β is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-β-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-β-mediated induction of myofibroblast differentiation and functional markers, such as α-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-β induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-β and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.

Species-Specific Fibroblasts Required for Triggering Invasiveness of Partially Transformed Oral Keratinocytes

This study tests the hypothesis that invasion of partially transformed keratinocytes is initiated by diffusible, proinvasive signals provided by species-specific fibroblasts. In vitro organotypic cultures of neoplastic human oral mucosa were constructed by growing a partially transformed, nontumorigenic keratinocytic cell line isolated from a dysplastic human oral lesion (DOK-ECACC94122104) on top of various types of connective tissue equivalents. Cultured tissues were analyzed by histomorphometry (depth and area of invasion: Dinv, Ainv) and immunohistochemistry. Presence of human fibroblasts in the matrix induced a local invasion of DOK (Dinv = 95.6 +/- 7.1 microm, Ainv = 45.8 +/- 3.5%). Minimal invasion (P < 0.05) was observed when DOK grew on simple collagen matrix (Dinv = 14.1 +/- 2.1 microm, Ainv = 3.7 +/- 0.8%) or matrices containing fibroblasts from mouse (Dinv = 11.5 +/- 4.0 microm, Ainv = 4.3 +/- 1.0%) or rat (Dinv = 15.6 +/- 1.2 microm, Ainv = 6.1 +/- 0.5%). In these cultures, local invasion could be induced by the presence of human fibroblasts in a bottom layer of the collagen matrix (P < 0.05) or by conditioned medium from organotypic cultures of DOK on human fibroblast-containing matrix (P < 0.05) but not by conditioned medium from human fibroblast monocultures (P > 0.05). Deposition of human collagen IV was observed at epithelial-matrix interface only when DOK behaved invasively. In conclusion, invasion of partially transformed oral keratinocytes was triggered by keratinocyte-induced fibroblast-derived diffusible factor(s) in a species-specific manner and associated with de novo synthesis of collagen IV.

Colon cancer cells: Pro-invasive signalling

Colon cancer results from erroneous renewal of the enteric epithelium. Mutations in stem cells, or their proliferative progenitors, cause accumulation of cells that invade into the stroma and continue to divide rather than migrating on top of the basement membrane prior to entering into apoptosis. Many of these changes in invasive activity appear to be related to the invasion-suppressor role of E-cadherin. We have also investigated Listeria monocytogenes and other enteric bacteria, since these bacteria stimulate invasion through the production of a β-casein-derived 13-amino acid peptide which is produced by enzymes present in the colon cancer ecosystem. The pro-invasive 13-amino acid peptide signals via small guanosine triphosphatases, which modulate the actin cytoskeleton, and via phosphorylation of the δ opioid receptor. The pro-invasive activity of this peptide is neutralized by the δ opioid receptor antagonist, naloxone. Since the δ opioid receptor belongs to the family of G protein-coupled receptors, implicated in colon cancer cell invasion signalling pathways, it is tempting to speculate that opioids could play a role in mediating this trait of malignant tumours.

Tenascin‐C and SF/HGF produced by myofibroblasts in vitro provide convergent proinvasive signals to human colon cancer cells through RhoA and Rac

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)-beta stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF-beta-upregulated extracellular matrix glycoprotein tenascin-C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast-stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)-like repeats of TNC through EGF-receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c-Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA-Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

Role of tissue stroma in cancer cell invasion.

Maintenance of epithelial tissues needs the stroma. When the epithelium changes, the stroma inevitably follows. In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. Stromal changes at the invasion front include the appearance of myofibroblasts, cells sharing characteristics with fibroblasts and smooth muscle cells. The main precursors of myofibroblasts are fibroblasts. The transdifferentiation of fibroblasts into myofibroblasts is modulated by cancer cell-derived cytokines, such as transforming growth factor-beta (TGF-beta). TGF-beta causes cancer progression through paracrine and autocrine effects. Paracrine effects of TGF-beta implicate stimulation of angiogenesis, escape from immunosurveillance and recruitment of myofibroblasts. Autocrine effects of TGF-beta in cancer cells with a functional TGF-beta receptor complex may be caused by a convergence between TGF-beta signalling and beta-catenin or activating Ras mutations. Experimental and clinical observations indicate that myofibroblasts produce pro-invasive signals. Such signals may also be implicated in cancer pain. N-Cadherin and its soluble form act as invasion-promoters. N-Cadherin is expressed in invasive cancer cells and in host cells such as myofibroblasts, neurons, smooth muscle cells, and endothelial cells. N-Cadherin-dependent heterotypic contacts may promote matrix invasion, perineural invasion, muscular invasion, and transendothelial migration; the extracellular, the juxtamembrane and the beta-catenin binding domain of N-cadherin are implicated in positive invasion signalling pathways. A better understanding of stromal contributions to cancer progression will likely increase our awareness of the importance of the combinatorial signals that support and promote growth, dedifferentiation, invasion, and ectopic survival and eventually result in the identification of new therapeutics targeting the stroma.

Parathyroid hormone may be a cancer promoter – an explanation for the decrease in cancer risk associated with ultraviolet light, calcium, and vitamin D

Epidemiological studies reporting an inverse association between sunlight exposure and risk for cancers of the breast, colon, and prostate, have not yet been explained. Since ultraviolet (UV) light promotes dermal vitamin D generation, studies suggesting that dietary calcium and vitamin D may likewise have cancer-preventive activity are potentially of relevance. UV light, calcium, and vitamin D have the common property of suppressing parathyroid hormone (PTH) production; these considerations raise the possibility that PTH may have promotional activity for certain cancers. PTH might function indirectly in this regard, by increasing hepatic production of the progression growth factor IGF-I, a likely cancer promoter. A more direct role is suggested by recent evidence that many cancers express receptors for PTH/PTH-related protein; these receptors mediate co-mitogenic and/or pro-invasive signals in some cancers. High risk for previous or concurrent neoplasms has been reported in patients with parathyroid adenomas. In light of the increase in cancer risk associated with hypertension, it is notable that PTH levels are typically increased in salt-sensitive hypertensives. Prospective case-control studies examining serum PTH in relation to subsequent cancer risk appear warranted.