Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

The pros and cons of targeting protein kinase C (PKC) in the management of cancer patients.

In the last years, PKC has become an attractive target for the treatment of cancer patients given its widely described role in carcinogenesis and tumor promotion. Despite the extensive research conducted on these phorbol ester receptors there is only limited knowledge about the contribution of each individual PKC isozyme in malignant transformation, mainly due to the different roles of each isozyme and their tissue-specificity. This diversity provides the unique opportunity to develop specific pharmacological agents, but the complex nature of the signaling pathways activated by different PKCs challenges selective drug therapies. Currently, several classes of PKC inhibitors including small molecule kinase inhibitors, biologic modulators, and anti-sense oligonucleotides are being evaluated for the treatment of different cancers where PKC isozymes were found to be deregulated as lung, colon, skin, prostate, and breast malignancies. In this article we will review which PKC isoforms are deregulated in different human cancers and summarize the mechanism of action of some of the major PKC modulators, analyzing the strengths and weaknesses of each one in the clinical setting.

New murine cell line derived from a spontaneous lung tumor induces paraneoplastic syndromes.

LP07 is a new cell line derived from P07 lung tumor, spontaneously arisen in a BALB/c mouse. LP07 is composed of heterogeneous epithelioid polyhedric cells that proliferate at a slow rate, have low plating efficiency and are unable to grow in soft agar. Only some LP07 cells expressed cytokeratins while most of them were positive for vimentin. Ultrastructure studies showed that LP07 cells established rudimentary intercellular unions, formed glandular-like conducts and presented conspicuous secretory granules, suggesting an epithelial-glandular origin, with neuroendocrine components. Upon injection LP07 cells formed poorly differentiated non-invasive adenocarcinomas, and tumor bearing mice developed leukocytosis, hypercalcemia and cachexia. This tumor cell line constitutes a useful tool to study lung tumor biology and paraneoplastic syndromes.

Involvement of fibronectin in the regulation of urokinase production and binding in murine mammary tumor cells.

Tumor invasion and metastasis development is a multistep process involving adhesion molecules as well as tumor proteases. It has been reported that tumor cells lacking fibronectin (FN) expression and engineered to re-express FN showed a marked reduction in metastatic ability. Besides its effects on cell adhesion and migration, FN could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we analyzed the production of urokinase-type plasminogen activator (uPA), and its receptor (uPAR), 2 molecules involved in the invasive phenotype, in cells over-expressing RGD wild-type FN (FNwt clones) or RGD-mutated FN (FN RGD-minus clones). Secreted uPA activity and antigen were significantly up-regulated in FN-expressing clones, although RGD-minus cells secreted approximately 50% less uPA than the FNwt ones. Interestingly, while control and FN RGD-minus clones were able to readily bind uPA to their surface, FNwt clones exhibited impaired uPA binding. Furthermore, treatment of the parental cell line as well as the control and FN-expressing clones with exogenous purified FN or RGD peptides induced up-regulation of uPA production and the reduction of uPA membrane binding, which was associated with lower expression of uPAR. This modulation by FN was found to be dependent on RGD sequence and beta1 integrin. These results strongly suggest a novel activity for the multifunctional glycoprotein FN regarding the regulation of uPA production as well as the capacity of tumor cells to bind uPA.

Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells.

Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40-fold lower levels of FN mRNA, only detectable by RT-PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220-bp proximal promoter region.