Gaddamanugu L. Prasad

Loss of expression of tropomyosin-1, a novel class II tumor suppressor that induces anoikis, in primary breast tumors

Suppression of tropomyosins (TMs), a family of actin-binding, microfilament-associated proteins, is a prominent feature of many transformed cells. Yet it is unclear whether downregulation of TMs occur in human tumors. We have investigated the expression of tropomyosin-1 (TM1) in human breast carcinoma tissues by in situ hybridization and immunofluorescence. TM1 mRNA and protein are readily detectable in normal mammary tissue. In contrast, TM1 expression is abolished in the primary human breast tumors. Expression of other TM isoforms, however, is variable among the tumors. The consistent and profound downregulation of TM1 suggests that TM1 may be a novel and useful biomarker of mammary neoplasms. These data also support the hypothesis that suppression of TM1 expression during the malignant conversion of mammary epithelium as a contributing factor of breast cancer. In support of this hypothesis, we show that the ability to suppress malignant growth properties of breast cancer cells is specific to TM1 isoform. Investigations into the mechanisms of TM1-induced tumor suppression reveal that TM1 induces anoikis (detachment induced apoptosis) in breast cancer cells. Downregulation of TM1 in breast tumors may destabilize microfilament architecture and confer resistance to anoikis, which facilitates survival of neoplastic cells outside the normal microenvironment and promote malignant growth.

Cytoskeletal organization in tropomyosin-mediated reversion of ras-transformation: Evidence for Rho kinase pathway

Tropomyosin (TM) family of cytoskeletal proteins is implicated in stabilizing actin microfilaments. Many TM isoforms, including tropomyosin-1 (TM1), are down-regulated in transformed cells. Previously we demonstrated that TM1 is a suppressor of the malignant transformation, and that TM1 reorganizes microfilaments in the transformed cells. To investigate how TM1 induces microfilament organization in transformed cells, we utilized ras-transformed NIH3T3 (DT) cells, and those transduced to express TM1, and/or TM2. Enhanced expression of TM1 alone, but not TM2, results in re-emergence of microfilaments; TM1, together with TM2 remarkably improves microfilament architecture. TM1 induced cytoskeletal reorganization involves an enhanced expression of caldesmon, but not vinculin, α-actinin, or gelsolin. In addition, TM1-induced cytoskeletal reorganization and the revertant phenotype appears to involve re-activation of RhoA controlled pathways in DT cells. RhoA expression, which is suppressed in DT cells, is significantly increased in TM1-expressing cells, without detectable changes in the expression of Rac or Cdc42. Furthermore, expression of a dominant negative Rho kinase, or treatment with Y-27632 disassembled microfilaments in normal NIH3T3 and in TM1 expressing cells. These data suggest that reactivation of Rho kinase directed pathways are critical for TM1-mediated microfilament assemblies.

A cDNA encoding a muscle-type tropomyosin cloned from a human epithelial cell line: Identity with human fibroblast tropomyosin TM1

Tropomyosins (TM) expressed by human epithelial cells have only recently been characterized, and no sequence data for them has appeared. We cloned a cDNA encoding a high molecular weight, muscle-type TM from a LS174T human colon carcinoma epithelial cell cDNA library. On sequence analysis this cDNA (TMe1) was virtually identical to the previously reported sequence for human fibroblast TM1 encoded by the hTM beta gene. Expression of TM1/TMe1 mRNA and protein are low in epithelial cells compared with fibroblasts. The results indicate that cells of different developmental lineages (entodermal and mesodermal) can produce identical TM beta gene splice products while regulating expression of those transcripts in a lineage-specific way.

Inhibition of Nuclear Accumulation of Phosphorylated ERK by Tropomyosin-1-Mediated Cytoskeletal Reorganization

AIM: Most neoplastic cells express diminished levels of tropomyosin (TM) family of actin-binding proteins, which leads to the formation of poorly organized cytoskeleton. The aberrant cytoskeleton is hypothesized to contribute to the neoplastic phenotype through deregulation of intracellular signaling. The aim of the study is to evaluate whether reorganization of cytoskeleton modulates signaling pathways. METHODS: We have utilized ras-transformed NIH3T3 (DT) cells and those transduced with TM1 (DT/TM1) as a model system. DT cells are highly malignant whereas the DT/TM1 cells contain reorganized cytoskeleton and exhibit revertant phenotype. Activation status of ras oncogene in DT and DT/TM1 cells was measured by GTP loading. Activation status and subcellular localization of extracellular signal regulated kinase (ERK) was measured in total, cytoplasmic and nuclear compartments by immunoblotting and confocal microscopy. RESULTS: Transduction of TM1 does not alter the activation status of oncogenic ras. Both parental DT and DT/TM1 cells exhibit similar levels of activated ERK in total cellular lysates, whereas DT/TM1 cells contain significantly less phosphorylated ERK (pERK) in the nuclear fraction. Disruption of cytosekeletal integrity results in increased nuclear content of pERK, suggesting that tropomyosin-1-induced microfilaments are critical for curtailing the nuclear accumulation of activated ERK, and may contribute to the anti-oncogenic effects of TM1. CONCLUSION: Our data suggest that spatiotemporal regulation of ERK by cytoskeleton is an important mechanism. Furthermore, aberrant microfilaments, present in neoplastic cells, fail to restrict nuclear localization of ERK, and hence contribute to deregulated ERK signaling.