To better understand the role of invadopodia and PDGFRα in metastasis, we utilized a subcutaneous tumor implantation model in which Twist1-expressing human breast tumor cells carrying shRNAs against PDGFRα or Tks5 were injected subcutaneously in nude mice. Knockdown of Tks5, an essential invadopodia component protein with no other known functions, allowed us to test whether invadopodia were required for Twist1-mediated metastasis. Knockdown of PDGFRα was used to determine whether PDGF signaling induced by Twist1 for invadopodia formation was required for metastasis. Although no significant differences in growth rate were observed, both Tks5 and PDGFRα knockdowns dramatically suppressed local invasion, with the primary tumors remaining relatively well-encapsulated in a layer of fibrotic tissue. In contrast, tumors expressing control knockdown constructs invaded through the local ECM, often as single cells. Furthermore, knockdown of both Tks5 and PDGFRα significantly reduced dissemination to the lungs, as measured by quantification of GFP-positive puncta in the lungs.
In microarray data sets of human breast cancer tumor samples, we found a strong correlation between Twist1 and PDGFRα expression, with PDGFRα consistently ranking within the top 1% of genes correlated with Twist1. Furthermore, in a Stage II breast cancer tissue array from the National Cancer Institute coexpression of Twist1 and PDGFRα was significantly associated with patient survival, indicating the importance of this pathway in human breast tumor progression.
Druggable targets regulating invadopodia formation and function
The connection between Twist1, PDGFR signaling, and invadopodia is exciting as it highlights several new therapeutic targets for targeting local invasion in the metastatic process. Namely, PDGFRα, Src, and metalloproteases localized in invadopodia are appealing, druggable targets for targeting invasion in breast cancer metastasis (see Figure ). As metastasis may occur via invadopodia-independent mechanisms in patient populations, Twist1 and PDGFRα coexpression may be appealing markers for patient stratification for treatment regimens targeting invadopodia.
Recent research in our lab revealed that Twist1 directly induces transcription of PDGFRα
PDGFRα and EGFR
As a direct target of Twist1 tightly associated with survival in human breast cancer patient tissue samples, PDGFRα is an especially appealing target for therapeutic intervention in breast cancer metastasis. The most well-known and studied PDGFR inhibitor is imatinib mesylate (Gleevec, Novartis), which also inhibits Abl and c-Kit tyrosine kinases[31
]. Data from clinical trials involving use of imatinib in advanced breast cancers has been discouraging with no clear objective responses[32
]. If PDGFR signaling is important for invasion and metastasis, however, improved survival in these patients with late stage disease would be unlikely as the cancer had already widely metastasized. Often severe gastrointestinal side effects of imatinib treatment also severely limited its utility in at least one trial[33
]. Another tyrosine kinase inhibitor, Sunitinib (Sutent, Pfizer), targets PDGFRs, VEGFs, Kit, RET, and CSF[33
]. Encouragingly, Sunitinib is much better tolerated and has had some effectiveness in preliminary clinical trials of metastatic breast cancer[34
]. The promiscuous inhibitory profile of Sunitinib makes it difficult to determine whether its effects on disease outcome are through inhibition of PDGFRs. In light of our discovery, it is important to examine patient tumor samples to determine whether Sunitinib suppresses invadopodia and local invasion. To truly understand the utility of these novel inhibitors in breast cancer, it will be necessary to identify patient populations that will respond best to the therapy. Development of more specific inhibitors that target only PDGFRs, including humanized monoclonal antibodies, may address some of the side effects due to off targeting.
Epidermal growth factor receptor (EGFR) signaling is also known to play an important role in regulation of invadopodia formation. The most characterized role of EGFR signaling in invadopodia is its function upstream of Src activation[35
]. HER-2 (human epidermal growth factor receptor 2) status is an important clinical marker for treatment, with about 30% of patients presenting increased levels of HER-2expression[33
]. Patients that are HER-2 positive are considered candidates for treatment with HER-2 inhibitors, including trastuzumab (Herceptin, Genentech) and lapatinib (Tykerb/Tyverb GSK)[36
]. Recently, new small molecule-based therapeutics targeting EGFR, including erlotinib (Tarceva, OSI Pharmaceuticals), have proven useful in other cancers with upregulation of EGFR signaling[37
]. OSI Pharmaceuticals investigated the properties of cancer cells resistant to EGFR inhibition and found that this subpopulation of cells displayed properties of EMT, including an increased dependence on PDGFR signaling[38
]. During EMT, PDGFR signaling may largely supplant or supplement the role of EGFR signaling in promoting invadopodia formation in breast cancer cells. This also suggests that the EMT process may play roles in not only mediating local invasion and metastasis, but also providing an escape mechanism from growth factor inhibition.
Src and its effectors
As the first proto-oncogene discovered, there is a large body of research focusing on not only the role of Src in cancer but also potential therapeutic interventions. It is generally recognized that Src plays multiple roles in carcinomas, promoting both proliferation and survival and driving invasion[40
]. The essential role of Src activation in invadopodia formation suggests that Src inhibitors should effectively prevent invadopodia formation and ECM degradation in tumors. Several Src inhibitors are already in the clinic and used to treat chronic myelogenous leukemia by virtue of their ability to also inhibit Abl kinase[41
]. Src activity is also upregulated in a wide variety of solid cancers, including colon, breast, gastric, and ovarian cancers[42
]. Several pharmaceutical companies have therefore developed Src kinase inhibitors with varying levels of success. Most Src inhibitors that have progressed to clinical trials in solid tumors (Dasatinib, Bristol-Meyers Squibb; Saracatinib, AstraZeneca; and Bosutinib, Wyeth) work by competitively binding the ATP-binding site of Src[40
]. Initial results from clinical trials of Src inhibitors in breast cancer have been mixed, with most single-agent trials resulting in no significant differences in survival or progression[43
]. Combination therapeutics have resulted in more positive, although modest, effects [42
].It is important to note, however, that all clinical trials regarding Src inhibitors in breast cancer have been conducted in unselected patient populations and the main readout for effectiveness has been tumor size and growth, not invasion. There are some indications, however, that patient stratification can predict responsiveness to Src kinase inhibition[44
]. Novel Src inhibitors targeting the peptide binding site of Src rather than the ATP-binding site (KX2-391, Keryx Biopharmaceuticals) may also prove to be more effective in solid tumors, although clinical trials involving these compounds are still preliminary and underway[45
Interestingly, a recent publication elucidated a detailed mechanisms for Src-kinase induced invadopodia formation[35
]. Rather than directly phosphorylating cortactin, Src instead activates the Abl-related non-receptor tyrosine kinase Arg that is responsible for cortactin phosphorylation. In this system, cortactin tyrosine phosphorylation is transiently required for cortactin-mediated actin polymerization in invadopodia. This is particularly interesting, as Gleevec, a drug often used to target PDGFR signaling, also inhibits Arg activity[31
]. The promiscuity of Gleevec could therefore target multiple levels of the signaling pathways regulating invadopodia formation, making it a promising target in selected patient populations[46
Several metalloproteases are enriched at invadopodia, including MMP2, MMP9, and MT1-MMP[47
]. The transmembrane metalloprotease MT1-MMP is essential for invadopodia proteolytic activity: knockdown of MT1-MMP in multiple cell lines almost completely eliminates associated matrix degradation[19
]. In addition, recent work has also elucidated the vital role of MT-MMPs in mediating invasion through three-dimensional matrices[49
].The central role of MT-MMPs in mediating extracellular proteolysis at invadopodia could be due to either its intrinsic collagenase/gelatinase activity or via activation of soluble MMPs by MT1-MMP[50
]. There is also evidence that hydroxymate metalloprotease inhibitors prevent not only ECM proteolysis, but also invadopodia formation through an unknown mechanism[19
As cancer cells must invade through both basement membranes and the ECM during metastasis, metalloproteases were quickly recognized as appealing targets to inhibit metastasis. Although results were promising in preclinical models, metalloprotease inhibitors have universally failed in clinical trials[51
]. Once again, clinical trials with metalloprotease inhibitors to date have invariably used unselected patient populations, often with late-stage disease. Additionally, early metalloprotease inhibitors were broad-spectrum inhibitors of multiple metalloproteases and often had acute toxicities that severely limited therapeutic doses[51
]. MMPs may also play anti-tumor functions in many tumors, as well. For example, MMP8-/- mice developed more papillomas upon carcinogen treatment[52
].In recent years, there has been a reemergence of interest in more targeted inhibition of metalloproteases. In particular, the fully human monoclonal antibody DX-2400 (Dyax Corp.) that targets MT1-MMP, has shown great promise in preclinical models in inhibiting invasiveness of cancer cell lines[53
]. In addition, a novel class of metalloprotease inhibitors, triple-helical transition state analogues, specifically targets the gelatinase and collagenase activities of metalloproteases (specifically MMP2 and 9)[54
]. Clinically addressing the role of metalloproteases in breast cancer metastasis will involve not only designing trials to maximize the impact of the therapeutics, but also finding novel inhibitors with greater specificity and fewer negative side-effects.