SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-ß Signaling Pathway Targeting the Type II Receptor.

Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-ß (TGF-ß) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-ß signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-ß signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-ß type II receptor (TßRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-ß signaling pathway. SPSB1 negatively regulates the TGF-ß signaling pathway through its interaction with both endogenous and overexpressed TßRII (and not TßRI) via its Spry domain. As such, TßRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TßRII at a low level by enhancing the ubiquitination levels and degradation rates of TßRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-ß signaling and migration and invasion of tumor cells.

Single live cell TGF-ß signalling imaging: breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-ß-Smad3 activity.

BACKGROUND: Metastasis is a process where only a small subset of cells is capable of successfully migrating to and propagating at secondary sites. TGF-ß signalling is widely known for its role in cancer metastasis and is associated with cell migration in whole cell populations. FINDINGS: We extend these findings by investigating the role of TGF-ß signalling in promoting migration and motility by imaging the signalling activity in live, individual MDA-MB-231 cancer cells utilizing a novel Smad3 Td-Tomato reporter adenovirus. Here we find that not all MDA-MB-231 cancer cells have similar TGF-ß mediated Smad3 transcription activity and display at least two distinct migratory populations. Importantly, Smad3 activity was significantly higher within migratory cells compared to non-migrated cells in wound healing and transwell assays. Furthermore, time-lapse experiments showed that MDA-MB-231 cells displaying Smad3 activity moved faster and a greater distance compared to cells not displaying Smad3 reporter activity. Interestingly, despite being more motile than cells with undetectable levels of Smad3 activity, high Smad3 activity was detrimental to cell motility compared to low and medium level of Smad3 activity. CONCLUSIONS: We have developed a method enabling real-time visualization of TGF-ß signalling in single live cells. Breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-ß-Smad3 activity. Those sub-populations may be responsible for tumor invasion and metastasis.

New reagents for improved in vitro and in vivo examination of TGF-ß signalling.

Transforming growth factor-ß (TGF-ß) signalling controls many aspects of cell behaviour and is implicated as a key regulator in tumour formation and progression. However, evaluating levels of active TGF-ß in culture medium or patient plasma and gaining definitive information regarding the activity of downstream substrates such as Sma- and Mad-related protein 3 (Smad3) in vivo with accuracy and sensitivity has been problematic. Therefore, to overcome these technical issues we have created a NIH3T3 cell line with stable pCAGA(12)-luc expression that can now be utilised to detect TGF-ß activity with high sensitivity. In addition, we have created an adenoviral Smad3 luciferase reporter construct pAd.CAGA(12)-luc to successfully infect cells for in vitro assays, or prior to injection into mice and used to measure transcriptional activity in vivo. Thus, the NIH3T3-pCAGA(12)-luc cell line and the pAd.CAGA(12)-luc adenovirus will be extremely useful tools to measure TGF-ß signalling activity with far greater efficiency and reliability compared to original and currently used reagents.

Sichuan pepper extracts block the PAK1/cyclin D1 pathway and the growth of NF1-deficient cancer xenograft in mice.

There is increasing evidence that more than 70% of cancers including pancreatic, breast and prostate cancers as well as neurofibromatosis (NF) are highly addicted to abnormal activation of the Ser/Thr kinase PAK1 for their growth. So far FK228 is the most potent among the HDAC (histone deacetylase) inhibitors that block the activation of both PAK1 and another kinase AKT, downstream of PI-3 kinase. However, FK228 is still in clinical trials (phase 2) for a variety of cancers (but not for NF as yet), and not available for most cancer/NF patients. Thus, we have been exploring an alternative which is already in the market, and therefore immediately useful for the treatment of those desperate cancer/NF patients. Here we provide the first evidence that extracts of Chinese/ Japanese peppercorns (Zanthoxyli Fructus) from the plant Zanthoxylum piperitum called "Hua Jiao"/"Sansho", block selectively the key kinase PAK1, leading to the downregulation of cyclin D1. Unlike FK228, these extracts do not inhibit AKT activation at the concentrations that block either cancer growth or PAK1 activation. The Chinese pepper extract selectively inhibits the growth of NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells, without affecting the growth of normal fibroblasts, and suppresses the growth of NF1-deficient human breast cancer (MDA-MB-231) xenograft in mice. Our data suggest that these peppercorn extracts would be potentially useful for the treatment of PAK1-dependent NF such as MPNST, in addition to a variety of PAK1-dependent cancers including breast cancers.

Hyperactivation of Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-beta signaling.

The latent transcription factor Stat3 is activated by gp130, the common receptor for the interleukin (IL)-6 cytokine family and other growth factor and cytokine receptors. Ligand-induced dimerization of gp130 leads to activation of the Stat1, Stat3 and Shp2-Ras-Erk signaling pathways. Here we assess genetically the contribution of exaggerated Stat3 activation to the phenotype of gp130 (Y757F/Y757F) mice, in which a knock-in mutation disrupts the negative feedback mechanism on gp130-dependent Stat signaling. Compared to gp130 (Y757F/Y757F) mice, reduced Stat3 activation in gp130 (Y757F/Y757F) Stat3(+/-) mice increased their lifespan, prevented splenomegaly, normalized exaggerated hepatic acute-phase response and lymphocyte trafficking, and suppressed the growth of spontaneously arising gastric adenomas in young mice. These lesions share histological features of gastric polyps in aging mice with monoallelic null mutations in Smad4, which encodes the common transducer for transforming growth factor (TGF)-beta signaling. Indeed, hyperactivation of Stat3 desensitizes gp130 (Y757F/Y757F) cells to the cytostatic effect of TGF-beta through transcriptional induction of inhibitory Smad7, thereby providing a novel link for cross-talk between Stat and Smad signaling in gastric homeostasis.

PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition.

Oncogenic RAS mutants such as v-Ha-RAS induce cell cycling, in particular the G1 to S transition, by upregulating cyclin D1 and downregulating p27, an inhibitor for cyclin-dependent kinases (CDKs). PI-3 kinase appears to be involved in the regulation of both cyclin D1 and p27. In this report, using two distinct inhibitors specific for PAK1-3 (CEP-1347 and WR-PAK18), we present the first evidence indicating that the PIX/Rac/CDC42-dependent Ser/Thr kinases PAK1-3, acting downstream of PI-3 kinase and upstream of the Raf/MEK/ERKs kinase cascade, is essential for RAS-induced upregulation of cyclin D1, but not downregulation of p27. Since these PAK-inhibitors block selectively the malignant growth of RAS transformants, in which PAK1 is constitutively activated, but not normal cell growth, it is suggested that RAS transformants are addicted to the high levels of PAK1 for their malignant entry to S phase.

Rho family-associated kinases PAK1 and rock.

Rho family GTPases (Rho, Rac and CDC42) share around 30% sequence identity with RAS family GTPases, and are essential for RAS-induced malignant transformation, i.e., aberrant serum/anchorage-independent growth and actin cytoskeleton-linked morphological changes. Oncogenic RAS mutants such as v-Ha-RAS trigger cell cycle entry (G0-G1 transition) mainly by up-regulating cyclin D1, an activator of cyclin-dependent kinases (CDK), and down-regulating p27, a CDK inhibitor. Although both Rac and CDC42 are clearly activated by RAS, there is so far no evidence that RAS activates Rho. In this chapter, we will discuss the role of these Rho family GTPases and their effectors, in particular the Ser/Thr kinases PAK1 and Rock, in RAS-induced serum/anchorage-independent cell cycling, and discuss several potential therapeutics, peptides or chemical compounds, that could block this oncogenic cell cycle signalling pathway.

The K252a derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth of RAS transformants.

BACKGROUND: Oncogenic RAS mutants such as v-Ha-RAS activate members of Rac/CDC42-dependent kinases (PAKs) and appear to contribute to the development of more than 30% of all human cancers. PAK1 activation is essential for oncogenic RAS transformation, and several chemical compounds that inhibit Tyr kinases essential for the RAS-induced activation of PAK1 strongly suppress RAS transformation either in cell culture or in vivo (nude mice). Although we have developed a cell-permeable PAK-specific peptide inhibitor called WR-PA18, so far no chemical (metabolically stable) compound has been developed that directly inhibits PAK1 in a highly selective manner. Thus, we have explored such a PAK1 inhibitor(s) among synthetic derivatives of an adenosine triphosphate antagonist. RESULTS: From the naturally occurring adenosine triphosphate antagonist K252a, we have developed two bulky derivatives, called CEP-1347 and KT D606 (a K252a dimer), which selectively inhibit PAKs or mixed-lineage kinases both in vitro and in cell culture and convert v-Ha-RAS-transformed NIH 3T3 cells to flat fibroblasts similar to the parental normal cells. Furthermore, these two K252a analogues suppress the proliferation of v-Ha-RAS transformants, but not the normal cells. CONCLUSION: These bulky adenosine triphosphate antagonists derived from K252a or related indolocarbazole compounds such as staurosporine would be potentially useful for the treatment of RAS/ PAK1-induced cancers, once their anti-PAK1 activity is significantly potentiated by a few additional chemical modifications at the sugar ring suggested in this paper.