Discovery of HSPG2 (Perlecan) as a Therapeutic Target in Triple Negative Breast Cancer.

In recent years, there have been significant advances in the treatment of breast cancer resulting in remarkably high survival rates. However, treatment options for metastatic triple negative breast cancer (TNBC) are quite limited due to a lack of identifiable, unique markers. Using a phage display-based whole cell biopanning procedure, we developed two human antibodies that bind to tumor cells with a metastatic TNBC phenotype. Our studies further identified domain 1 of HSPG2 (perlecan) protein as the cognate cell surface antigen bound by the antibody. Immunohistochemistry studies utilizing patient tissue samples revealed significant cell surface expression of HSPG2 in both primary tumors and metastatic lesions. Further, higher HSPG2 expression correlated with poor survival in TNBC. The affinity-matured antibody inhibited the growth of triple negative MDA-MB-231 tumors to a greater extent in nude mice than in NSG mice, pointing to the potential role of natural killer cell-mediated antibody-dependent cell cytotoxicity. This mechanism of action was confirmed through in vitro assays using mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). These results suggest that HSPG2 is a promising target in metastatic TNBC and HSPG2-targeted antibodies could represent a potentially novel class of targeted therapeutics for TNBC.

Breast cancer cell-derived fibroblast growth factors enhance osteoclast activity and contribute to the formation of metastatic lesions.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been implicated in promoting breast cancer growth and progression. While the autocrine effects of FGFR activation in tumor cells have been extensively studied, little is known about the effects of tumor cell-derived FGFs on cells in the microenvironment. Because FGF signaling has been implicated in the regulation of bone formation and osteoclast differentiation, we hypothesized that tumor cell-derived FGFs are capable of modulating osteoclast function and contributing to growth of metastatic lesions in the bone. Initial studies examining FGFR expression during osteoclast differentiation revealed increased expression of FGFR1 in osteoclasts during differentiation. Therefore, studies were performed to determine whether tumor cell-derived FGFs are capable of promoting osteoclast differentiation and activity. Using both non-transformed and transformed cell lines, we demonstrate that breast cancer cells express a number of FGF ligands that are known to activate FGFR1. Furthermore our results demonstrate that inhibition of FGFR activity using the clinically relevant inhibitor BGJ398 leads to reduced osteoclast differentiation and activity in vitro. Treatment of mice injected with tumor cells into the femurs with BGJ398 leads to reduced osteoclast activity and bone destruction. Together, these studies demonstrate that tumor cell-derived FGFs enhance osteoclast function and contribute to the formation of metastatic lesions in breast cancer.

EMT reversal in human cancer cells after IR knockdown in hyperinsulinemic mice.

Type 2 diabetes (T2D) is associated with increased cancer risk and cancer-related mortality. Data herein show that we generated an immunodeficient hyperinsulinemic mouse by crossing the Rag1(-/-) mice, which have no mature B or T lymphocytes, with the MKR mouse model of T2D to generate the Rag1(-/-) (Rag/WT) and Rag1(-/-)/MKR(+/+) (Rag/MKR) mice. The female Rag/MKR mice are insulin resistant and have significantly higher nonfasting plasma insulin levels compared with the Rag/WT controls. Therefore, we used these Rag/MKR mice to investigate the role of endogenous hyperinsulinemia on human cancer progression. In this study, we show that hyperinsulinemia in the Rag/MKR mice increases the expression of mesenchymal transcription factors, TWIST1 and ZEB1, and increases the expression of the angiogenesis marker, vascular endothelial growth factor A (VEGFA). We also show that silencing the insulin receptor (IR) in the human LCC6 cancer cells leads to decreased tumor growth and metastases, suppression of mesenchymal markers vimentin, SLUG, TWIST1 and ZEB1, suppression of angiogenesis markers, VEGFA and VEGFD, and re-expression of the epithelial marker, E-cadherin. The data in this paper demonstrate that IR knockdown in primary tumors partially reverses the growth-promoting effects of hyperinsulinemia as well as highlighting the importance of the insulin receptor signaling pathway in cancer progression, and more specifically in epithelial-mesenchymal transition.

IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas.

Patients with metastatic or recurrent and refractory sarcomas have a dismal prognosis. Therefore, new targeted therapies are urgently needed. This study was designed to evaluate chimeric antigen receptor (CAR) T cells targeting the type I insulin-like growth factor receptor (IGF1R) or tyrosine kinase-like orphan receptor 1 (ROR1) molecules for their therapeutic potential against sarcomas. Here, we report that IGF1R (15/15) and ROR1 (11/15) were highly expressed in sarcoma cell lines including Ewing sarcoma, osteosarcoma, alveolar or embryonal rhabdomyosarcoma, and fibrosarcoma. IGF1R and ROR1 CAR T cells derived from eight healthy donors using the Sleeping Beauty (SB) transposon system were cytotoxic against sarcoma cells and produced high levels of IFN-γ, TNF-α and IL-13 in an antigen-specific manner. IGF1R and ROR1 CAR T cells generated from three sarcoma patients released significant amounts of IFN-γ in response to sarcoma stimulation. The adoptive transfer of IGF1R and ROR1 CAR T cells derived from a sarcoma patient significantly reduced tumor growth in pre-established, systemically disseminated and localized osteosarcoma xenograft models in NSG mice. Infusion of IGF1R and ROR1 CAR T cells also prolonged animal survival in a localized sarcoma model using NOD/scid mice. Our data indicate that both IGF1R and ROR1 can be effectively targeted by SB modified CAR T cells and that such CAR T cells may be useful in the treatment of high risk sarcoma patients.

SWIFT MRI enhances detection of breast cancer metastasis to the lung.

PURPOSE: To evaluate the capability of longitudinal MR scans using sweep imaging with Fourier transformation (SWIFT) to detect breast cancer metastasis to the lung in mice. METHODS: Mice with breast cancer metastatic to the lung were generated by tail vein injection of MDA-MB-231-LM2 cells. Thereafter, MR imaging was performed every week using three different pulse sequences: SWIFT [echo time (TE) ∼3 µs], concurrent dephasing and excitation (CODE; TE ∼300 µs), and three-dimensional (3D) gradient echo (GRE; TE = 2.2 ms). Motion during the long SWIFT MR scans was compensated for by rigid-body motion correction. Maximum intensity projection (MIP) images were generated to visualize changes in lung vascular structures during the development and growth of metastases. RESULTS: SWIFT MRI was more sensitive to signals from the lung parenchyma than CODE or 3D GRE MRI. Metastatic tumor growth in the lungs induced a progressive increase in intensity of parenchymal signals in SWIFT images. MIP images from SWIFT clearly visualized lung vascular structures and their disruption due to progression of breast cancer metastases in the lung. CONCLUSION: SWIFT MRI's sensitivity to fast-decaying signals and tolerance of magnetic susceptibility enhances its effectiveness at detecting structural changes in lung parenchyma and vasculature due to breast cancer metastases in the lung.

Acquired resistance to tamoxifen is associated with loss of the type I insulin-like growth factor receptor: implications for breast cancer treatment.

The role of the insulin-like growth factor (IGF) system in breast cancer is well defined, and inhibitors of this pathway are currently in clinical trials. The majority of anti-IGF1R clinical trials are in estrogen receptor-positive patients who have progressed on prior endocrine therapy; early reports show no benefit for addition of IGF1R inhibitors to endocrine therapy in this setting. In this study, we examined the effectiveness of IGF1R inhibitors in vitro by generating tamoxifen-resistant (TamR) cells. We found that TamR cells had diminished levels of IGF1R with unchanged levels of insulin receptor (IR), and failed to respond to IGF-I-induced Akt activation, proliferation, and anchorage-independent growth while retaining responsiveness to both insulin and IGF-II. The IGF1R antibody dalotuzumab inhibited IGF-I-mediated Akt phosphorylation, proliferation, and anchorage-independent growth in parental cells, but had no effect on TamR cells. An IGF1R tyrosine kinase inhibitor, AEW541, with equal potency for the IGF1R and IR, inhibited IGF-I-, IGF-II-, and insulin-stimulated Akt phosphorylation, proliferation, and anchorage-independent growth in parental cells. Interestingly, AEW541 also inhibited insulin- and IGF-II-stimulated effects in TamR cells. Tamoxifen-treated xenografts also had reduced levels of IGF1R, and dalotuzumab did not enhance the effect of tamoxifen. We conclude that cells selected for tamoxifen resistance in vitro have downregulated IGF1R making antibodies directed against this receptor ineffective. Inhibition of IR may be necessary to manage tamoxifen-resistant breast cancer.

Targeting the type I insulin-like growth factor system for breast cancer therapy.

The insulin-like growth factors (IGFs) acting via the type I IGF receptor (IGF-1R) regulate cancer cell proliferation, survival, metabolism and metastasis. Drugs targeting the IGF-1R are being tested in human clinical trials for cancer therapy and it seems likely that this class of drugs could be approved soon. Recent data suggests that insulin receptor, which is closely related to IGF-1R, should also be targeted to maximally inhibit the system. Furthermore, biomarkers that will identify patients whose tumors are driven by IGF-1R and biomarkers that allow monitoring or prediction of response are needed. This article reviews the different drugs against IGF-1R that are being tested in and how this receptor pathway can be optimally targeted for cancer therapy with an emphasis on breast cancer therapy.

A novel reduced immunogenicity bispecific targeted toxin simultaneously recognizing human epidermal growth factor and interleukin-4 receptors in a mouse model of metastatic breast carcinoma.

PURPOSE: To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE(38)KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo. EXPERIMENTAL DESIGN: A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE(38)). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231. RESULTS: EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by approximately 90% with no apparent loss in in vitro or in vivo activity. CONCLUSIONS: Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials.

Sequencing of type I insulin-like growth factor receptor inhibition affects chemotherapy response in vitro and in vivo.

PURPOSE: The aim of this study was to determine the optimal sequence of combining anti-type I insulin-like growth factor receptor (IGF1R) antibodies with chemotherapeutic drugs in cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: MCF-7 and LCC6 cells were treated with subcytotoxic concentrations of doxorubicin with or without anti-IGF1R antibodies (scFv-Fc or EM164 and its humanized version AVE1642). Treatments were given simultaneously, doxorubicin followed by anti-IGF1R antibody, or anti-IGF1R antibody followed by doxorubicin, with measurement of in vitro proliferation, apoptosis, and anchorage-independent growth. The effects of sequencing on LCC6 xenograft growth and metastasis were studied. RESULTS: Doxorubicin followed by anti-IGF1R antibody (scFv-Fc or EM164) was the most effective combination strategy to inhibit cell monolayer growth and anchorage-independent growth. This sequential combination triggered increased poly (ADP-ribose) polymerase cleavage compared with other treatment sequences. The reverse sequence, antibody followed by doxorubicin treatment, protected cells from chemotherapy by decreasing apoptosis, arresting cells in S phase, and inhibiting the level and activity of topoisomerase IIalpha. Finally, our in vivo data show that recovery of IGF1R prior to doxorubicin therapy resulted in the best therapeutic responses. Low doses of AVE1642 that allowed IGF1R expression to recover at one week were more effective in combination with doxorubicin than higher antibody doses. CONCLUSION: The timing of IGF1R inhibition affects responses to chemotherapy. The optimal sequence was doxorubicin followed by anti-IGF1R antibody, whereas the opposite sequence inhibited doxorubicin effects. Thus, the dose and sequencing of anti-IGF1R therapies should be considered in the design of future clinical trials.

Detection and downregulation of type I IGF receptor expression by antibody-conjugated quantum dots in breast cancer cells.

The type I insulin-like growth factor (IGF) receptor (IGF1R) is a transmembrane tyrosine kinase involved in breast cancer proliferation, survival, and metastasis. Several monoclonal antibodies directed against the receptor are in clinical trials. In order to develop a methodology to detect and measure IGF1R levels in breast cancer cells, we covalently conjugated an IGF1R antibody, AVE-1642, with quantum dots (Qdots), which are nanocrystals that emit fluorescence upon excitation. AVE-1642 Qdots only bound to cells that express IGF1R, and measured IGF1R levels by fluorescence emission at 655 nm. After binding to the cell surface, AVE-1642 Qdots underwent receptor mediated endocytosis, localized to endosome, and later translocated into the nucleus. Treating MCF-7 cells with AVE-1642 Qdots, but not unconjugated Qdots alone, downregulated IGF1R levels and rendered cells refractory to IGF-I stimulation. Furthermore, cell proliferation was slightly inhibited by AVE-1642 Qdots, but not the unconjugated Qdots. Our data suggest that AVE-1642 Qdots can be used to detect IGF1R expression and measure changes in cell surface receptor levels. In addition, the inhibitory effect of AVE-1642 Qdots to cell proliferation implies that it may serve as a traceable therapeutic agent.

Regulation of breast cancer metastasis by IGF signaling.

The insulin-like growth factors (IGFs) signaling via the type I insulin-like growth factor receptor (IGF-1R) regulate multiple aspects of malignancy. The importance of IGF-1R in regulating the malignant phenotype is currently being validated in numerous clinical trials for cancer including breast cancer. This review discusses the regulation of breast cancer metastasis by IGF-1R. IGF-1R stimulates invasion and survival in anchorage independent conditions. The regulation of metastasis independently of tumor growth by IGF-1R is also discussed. Finally, the impact of this on clinical trial design and outcomes, and the need for biomarkers, other than reduction in tumor size, are discussed in light of the fact that inhibition of metastasis is not measured in conventional clinical trial design.

Drug evaluation: CP-751871, a human antibody against type I insulin-like growth factor receptor for the potential treatment of cancer.

Several phase II trials of CP-751871 are currently underway, including a phase Ib/II [corrected] trial of CP-751871 in combination with paclitaxel and carboplatin in patients with advanced NSCLC, a phase II trial of CP-751871 in combination with docetaxel and prednisone in patients with hormone-refractory prostate cancer, and a phase II trial of CP-751871 in combination with exemestane in hormone receptor positive advanced breast cancer [corrected]

Disrupting insulin-like growth factor signaling as a potential cancer therapy.

The type I insulin-like growth factor receptor (IGF-IR) plays multiple roles in several cancers and increased circulating levels of insulin-like growth factor-I (IGF-I) are associated with increased risk of breast, colon, and prostate cancers. Because IGF-II and insulin signal via the insulin receptor (IR) to stimulate the growth of cancer cells, inhibition of IR might be necessary to totally disrupt the action of IGFs and their receptors. This review describes the well-recognized roles of IGF-IR in driving the malignant phenotype, examines the evidence that perhaps IR should also be targeted to inhibit the effects of the IGF ligands and insulin in cancer, describes the strategies to disrupt IGF signaling in cancer, and highlights some key issues that need to be considered as clinical trials targeting IGF-IR proceed.

Inhibitors of insulin-like growth factor signaling: a therapeutic approach for breast cancer.

The peptide growth factors IGF-I and IGF-II not only play a role in the development of the mammary gland but are also implicated in breast cancer. Several reagents disrupting IGF signaling have been developed and clinical trials validating IGF signaling as a target in cancer therapy are underway. This review highlights the approaches to inhibiting IGF signaling in breast cancer.

Down-regulation of insulin receptor by antibodies against the type I insulin-like growth factor receptor: implications for anti-insulin-like growth factor therapy in breast cancer.

Insulin-like growth factor-I (IGF-I), IGF-II, and insulin have all been implicated in regulating several aspects of the malignant phenotype via the type I IGF receptor (IGF1R) and insulin receptor (IR). We have previously shown that a chimeric single-chain antibody against IGF1R (scFv-Fc) and a murine antibody EM164 down-regulate IGF1R, making breast cancer cells unresponsive to IGF-I. To determine if IR signaling is affected, we examined regulation of IR in MCF-7 cells after exposure to these antibodies. Surprisingly, both scFv-Fc and EM164 resulted in decreased levels of IR in vitro and in vivo despite their lack of reactivity against IR. Twenty-four-hour pretreatment with EM164 also inhibited insulin-mediated phosphorylation of IR and insulin-stimulated proliferation of MCF-7 cells. Neither scFv-Fc nor EM164 caused down-regulation of IR in cells that express very low levels of IGF1R or no IGF1R. Expression of IGF1R was required for IR down-regulation, which was specific as neither antibody caused down-regulation of beta1 integrin or epidermal growth factor receptor. Reagents that disrupt lipid rafts inhibited IR down-regulation by the antibodies, suggesting that IR in close physical proximity to IGF1R in lipid rafts was being endocytosed. Our data show that down-regulation of IR by monoclonal antibodies against IGF1R requires the coexpression of IGF1R and may be due to endocytosis of hybrid IR/IGF1R or holo-IR. Thus, antibodies against IGF1R provide inhibition of both IGF and insulin signaling in cancer cells.

Motility response to insulin-like growth factor-I (IGF-I) in MCF-7 cells is associated with IRS-2 activation and integrin expression.

In MCF-7L cells, insulin-like growth factor-I (IGF-I) stimulates activation of insulin receptor substrate-1 (IRS-1) and enhances cell proliferation. While others have shown that IGF-I enhances cell motility in MCF-7 cells, we have not been able to demonstrate this. To determine if the source of MCF-7 cells account for these reported differences, we examined the MCF-7 cells available from the American Type Culture Collection (MCF-7/ATCC) and compared them to the MCF-7L cells maintained in our laboratory. Both MCF-7L and MCF-7/ATCC grew in response to 5 nM IGF-I and 1 nM estradiol. However, only MCF-7/ATCC demonstrated IGF-I stimulated motility. Immunoprecipitation of IRS substrates followed by anti-phosphotyrosine blotting demonstrated that both IRS-1 and IRS-2 were activated by IGF-I in these cells. However, MCF-7/ATCC cells had greater phosphorylation of IRS-2 compared to MCF-7L. Immunoblots showed that levels of IRS-1 and IRS-2 were comparable between cell lines. We have previously shown that fibronectin-binding integrins are necessary for IGF-stimulated motility. Similar levels of beta1 integrin were detected in both strains of MCF-7. However, low levels of alpha5 and alpha3 were detected in MCF-7L cells whereas high levels of alpha3 and alpha5 integrin were expressed in MCF-7/ATCC cells. Inhibition of integrin function by a blocking antibody or inhibitory peptide diminished IGF-mediated motility in MCF-7/ATCC. In MCF-7/ATCC cells, IGF-I stimulation was associated with a movement of IRS-2 to the leading edge of filopodia. Thus, patterns of integrin expression among breast cancer cell lines may partially explain the different motility behavior of cells in response to IGF-I. IRS-2 activation and integrin occupancy are both required for IGF-stimulated motility.

A dominant negative type I insulin-like growth factor receptor inhibits metastasis of human cancer cells.

We have previously shown that LCC6 wild-type (WT) cells, a metastatic variant of MDA-MB-435 cancer cells originally derived from a breast cancer patient, exhibit enhanced motility in response to IGF-I compared with the parent MDA-MB-435 cells. To further understand the role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in cancer metastasis we inhibited signaling via IGF1R using a C-terminal-truncated IGF1R. The truncated receptor retains the ligand binding domain but lacks the autophosphorylated tyrosine residues in the carboxyl terminus. Cells stably transfected with this truncated receptor (LCC6-DN cells) overexpressed the truncated IGF1R messenger RNA nearly 50-fold over endogenous receptor. The truncated receptor in the LCC6-DN cells behaved in a dominant negative manner to inhibit endogenous IGF1R activation by IGF-I. Compared with the LCC6-WT cells, LCC6-DN cells failed to phosphorylate the adaptor proteins insulin receptor substrate-1 and -2 in response to IGF-I and did not activate Akt after exposure to IGF-I. Unlike LCC6-WT cells, LCC6-DN cells did not show enhanced motility in response to IGF-I. To assay for metastasis, LCC6-WT and LCC6-DN cells were injected into the mammary fat pads of mice, and the primary xenograft tumors were removed after 21 days. Mice sacrificed 5 weeks later showed multiple lung metastases derived from LCC-WT xenografts, whereas mice harboring LCC6-DN xenografts showed no lung metastases. Our data show that IGF1R can regulate several aspects of the malignant phenotype. In these cells, metastasis but not proliferation requires IGF1R function.

A chimeric humanized single-chain antibody against the type I insulin-like growth factor (IGF) receptor renders breast cancer cells refractory to the mitogenic effects of IGF-I.

Insulin-like growth factors (IGFs) stimulate breast cancer proliferation, motility, and survival. The type I IGF receptor (IGF1R) mediates the effects of IGF-I. Thus, inhibition of IGF1R activation could inhibit IGF action in breast cancer cells. A single-chain antibody directed against IGF1R (IGF1R scFv-Fc) has been shown to partially inhibit xenograft growth of MCF-7 cells in athymic mice. In this study, we have examined the effects of scFv-Fc on IGF1R signaling in the estrogen receptor-positive (ER+) MCF-7 breast cancer cells in vitro and in vivo. The antibody stimulated IGF1R activation in vitro in MCF-7 cells and was unable to block IGF-I effects. The antibody also stimulated proliferation of MCF-7 cells in monolayer growth assays. To determine how scFv-Fc could stimulate in vitro growth yet inhibit in vivo tumor growth, we examined the effect of scFv-Fc on IGF1R expression. In MCF-7 cells, scFv-Fc down-regulated IGF1R levels after 2 h, and the levels were greatly reduced after 24 h. In contrast, IGF-I treatment over the same time period did not affect IGF1R levels. Twenty-four-h pretreatment of cells with scFv-Fc blocked IGF-I mediated phosphorylation of insulin receptor substrate-1 and subsequent extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 and phosphatidylinositol 3'-kinase activation. In contrast, cells treated with 5 nM IGF-I for 24 h still retained the ability to further activate downstream signaling pathways in response to IGF-I. Moreover, pretreatment of MCF-7 cells with scFv-Fc rendered them refractory to further proliferation induced by additional IGF-I. Twenty-four-h pretreatment of cells with scFv-Fc also inhibited IGF-I stimulated anchorage-independent growth. scFv-Fc did not enhance antibody-dependent cell-mediated cytotoxicity. In vivo, treatment of mice bearing MCF-7 xenograft tumors with scFv-Fc resulted in near complete down-regulation of IGF1R. Our data show that scFv-Fc stimulates biochemical activation of IGF1R, then causes receptor down-regulation, making MCF-7 cells refractory to additional IGF-I exposure. These results indicate that such chimeric single-chain antibodies against IGF1R have future potential in breast cancer therapy by causing down-regulation of receptor.

New mechanisms of signal transduction inhibitor action: receptor tyrosine kinase down-regulation and blockade of signal transactivation.

The explosion of signal transduction research over the last 10 years has provided a unique insight into the complexity of these intricate pathways. Whereas intermediates of multiple signaling pathways have been identified, understanding their function and, in particular, the interactions between them has become a daunting task. The increasing evidence that many of these pathways can cross-talk with each other via signal transactivation inevitably raises the question of how cells determine specificity in signaling. Despite the mind-numbing complexity of these pathways, progress has been made in developing highly specific and potent signal transduction inhibitors (STIs). STIs show promise in the treatment of cancer in preclinical studies and are currently in a number of clinical trials. Whereas many of these agents were "rationally designed," we barely understand their mechanisms of action. This review will highlight how recent studies using these STIs have elucidated novel mechanisms of STI action that may be used in the development of new therapeutic strategies for the treatment of cancer.