Andres J. Klein-Szanto

The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis

MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently and still remains largely unexplored. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.

Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

Biomechanical Remodeling of the Microenvironment by Stromal Caveolin-1 Favors Tumor Invasion and Metastasis

Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.

AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth

Activation of the PI3K/AKT pathway may contribute to tumorigenesis. AKT mediates survival signals that protect cells from apoptosis and, thus, is a potentially important therapeutic target. To determine the frequency of AKT activation in human ovarian cancer, we screened a tumor tissue microarray with a phospho-specific pan-AKT (Ser473) antibody, which revealed elevated staining in 21 of 31 (68%) ovarian carcinomas. Phospho-AKT staining was associated with that of phospho (active)-mTOR in 27 of 31 (87%) ovarian tumors, with 17 (55%) tumors showing elevated phospho-mTOR positivity. We tested the effects of AKT/mTOR activation on the therapeutic sensitivity of ovarian cancer cells. Pretreatment of SKOV3 cells, which exhibit constitutive AKT activity under low serum conditions, with the PI3K inhibitor LY294002 augmented cisplatin-induced apoptosis. In contrast, ovarian cancer cell lines OVCAR4 and OVCAR5, which have low basal levels of AKT activity, did not show increased cisplatin-induced apoptosis when pretreated with LY294002. In addition, inhibition of mTOR activity with rapamycin resulted in G1 arrest in SKOV3 cells, but not in OVCAR4 or OVCAR5 cells. Collectively, these findings indicate that active AKT and downstream mTOR represent potentially important therapeutic and/or chemopreventive targets in ovarian cancer.

Increased expression of stem cell markers in malignant melanoma.

The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of melanocytic stem cells have been described recently. CD166 is expressed on the surface of mesenchymal stem cells and has been found on human melanoma cell lines. CD133 is expressed on the surface of dermal-derived stem cells that are capable of differentiating into neural cells. Nestin is an intermediate filament expressed in the cytoplasm of neuroepithelial stem cells. In this study, we evaluate the expression of these markers and possible differences among banal nevi, primary melanoma, and metastastic melanoma. Tissue microarrays containing normal tissue and 226 melanocytic lesions (71 banal nevi, 71 in situ and invasive melanomas, and 84 metastatic melanomas) were studied by immunohistochemistry using monoclonal antibodies CD166, CD133, and nestin. A significantly greater percentage of melanomas (combined primary and metastatic) contained cells that expressed CD166 (P=0.005), CD133 (P=0.003), and nestin (P=0.03) than banal nevi. Only nestin showed a statistical difference when comparing primary and metastatic melanoma (P=0.05). A stepwise increase in the proportion of lesions expressing all three markers was observed from banal nevi (2/19) to primary melanomas (8/17) to metastatic melanoma (19/28), P=0.0005. All cases of metastatic melanoma expressed at least one stem cell marker. The increased expression of CD166, CD133, and nestin in melanoma suggests that progression to malignant melanoma likely involves genetic pathways instrumental to stem cell biology and normal tissue development. Further studies and characterization of these pathways may also reveal new prognostic markers for a disease whose prognosis in advanced stages is dismal.

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial‐specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic‐free Ca2+ [Ca2+]c. In the present study we show that the mitochondria‐to‐nucleus stress signaling induces invasive phenotypes in otherwise non‐invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor‐specific markers cathepsin L and transforming growth factor β (TGFβ) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4‐ to 6‐fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca2+‐dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with ∼70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.

Nipple aspirate fluid: a promising non-invasive method to identify cellular markers of breast cancer risk

To evaluate the feasibility of nipple aspiration and to identify intermediate markers of breast cancer risk, nipple aspirate fluid (NAF) was collected from 177 subjects using a modified breast pump. The first 33 subjects demonstrated that we could obtain NAF quickly, reliably and repeatedly. Specimens from the remaining 144 subjects were collected to evaluate promising cellular biomarkers. NAF was obtained in 167 out of 177 (94%) subjects overall and in 99% of the 144 most recent subjects. Sufficient NAF was obtained to evaluate cytology in 160 out of 167 (96%) cases and specimens were sufficiently cellular to analyse DNA markers in 53% of cases. Among the last 144 subjects, menopausal status did not influence the ability to obtain NAF. NAF cytology correlated with increased breast cancer risk (P = 0.002). Using computerized image analysis of NAF epithelial cells, DNA index (P = 0.0002), percentage of cells in G2M (P = 0.05) and percentage of cells with hypertetraploidy (P = 0.002) increased as cytology became more abnormal. Our data indicate that NAF can be obtained in essentially all eligible subjects; that breast epithelial cells are evaluable in > 95% of NAF samples for cytology and in over half of NAF samples for DNA index (ploidy) and cell cycle analysis; and that abnormal NAF cytology correlates with increased breast cancer risk. This suggests that biomarkers identified in nipple aspirate fluid may prove useful either as an adjunct to currently accepted breast cancer screening methods, or to evaluate response to a chemopreventive agent.

Clinical Implications of Fibroblast Activation Protein in Patients with Colon Cancer

Purpose: Human fibroblast activation protein (FAP)/seprase is a 97-kDa surface glycoprotein expressed on tumor associated fibroblasts in the majority of epithelial cancers including colon adenocarcinomas. FAP overexpression in human tumor cells has been shown to promote tumor growth in animal models, and clinical trials targeting FAP enzymatic activity have been initiated. The primary objective of this study was to evaluate the clinical significance of stromal FAP in human colon cancers by immunohistochemisty. Experimental Design: Sections of paraffin-embedded resected primary human colon cancer specimens from 1996 through 2001 within the Fox Chase Cancer Center tumor bank were stained with D8 antibody directed against FAP/seprase. Xenotransplanted human colorectal tumors in mice were examined similarly for stromal FAP in tumors of different sizes. Overall percentage of stromal FAP staining of the primary tumor was assessed semiquantitatively (0, 1+, 2+, 3+) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. Results: One hundred thirty-eight patients with resected specimens were available for study (mean follow-up, 1,050 days) with 6 (4%) stage I, 52 (38%) stage II, 43 (31%) stage III, and 37 (27%) stage IV patients. FAP was detected in >93% of specimens. Semiquantitative staining was scored as 1+ in 28 (20%), 2+ in 52 (38%), and 3+ in 49 (35%). FAP staining intensity was graded as weak in 45 (33%), intermediate in 48 (35%), and dark in 36 (26%). Stromal FAP was found to correlate inversely with tumor stage (semiquantitative, P = 0.01; intensity, P = 0.009) and with tumor size of the tumor xenograft model (correlation coefficient, −0.61; P = 0.047), suggesting that stromal FAP may have a greater role in the early development of tumors. Furthermore, greater stromal FAP for patients with known metastatic disease was associated with a decreased survival. Conclusion: Our data indicate that patients whose colon tumors have high levels of stromal FAP are more likely to have aggressive disease progression and potential development of metastases or recurrence. This study affirms the rationale for ongoing clinical investigations using FAP as a therapeutic target to disrupt FAP-driven tumor progression in patients with metastatic disease. It also suggests that the effects of FAP inhibition should be investigated in earlier-stage tumors, given its high levels and potential effect earlier in the course of the disease.

Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin–ezrin–radixin family of cytoskeleton–associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression

Metastasis is one of the most important and complex processes in human neoplastic disease. A large number of both positive and negative events must occur to permit a tumor cell to colonize a distant site successfully. To identify mouse strains that harbor dominant genetic modifiers of this process, a strain survey was initiated utilizing a transgenic mouse mammary tumor model that exhibits a high incidence of pulmonary metastases. The transgenic animal was bred to 27 different inbred strains of mice and scored for the metastatic organ tropism and metastatic density. Thirteen strains were identified that had a statistically significant reduction in the numbers of pulmonary metastases. In addition, 10 strains were identified that altered the kinetics of induction of the primary mammary tumor. These strains will likely provide useful model systems for the analysis of genetic interactions in the initiation and progression of mammary adenocarcinomas. Int. J. Cancer 77:640–644, 1998.