Insulin receptor substrates (IRSs) are signaling adaptors that play a major role in the metabolic and mitogenic actions of insulin and insulin-like growth factors. Reports have recently noted increased levels, or activity, of IRSs in many human cancers, and some have linked this to poor patient prognosis. We found that overexpressed IRS-1 was constitutively phosphorylated in vitro and in vivo and that transgenic mice overexpressing IRS-1 or IRS-2 in the mammary gland showed progressive mammary hyperplasia, tumorigenesis, and metastasis. Tumors showed extensive squamous differentiation, a phenotype commonly seen with activation of the canonical ␤-catenin signaling pathway. Consistent with this, IRSs were found to bind ␤-catenin in vitro and in vivo. IRS-induced tumorigenesis is unique, given that the IRSs are signaling adaptors with no intrinsic kinase activity, and this supports a growing literature indicating a role for IRSs in cancer. This study defines IRSs as oncogene proteins in vivo and provides new models to develop inhibitors against IRSs for anticancer therapy.Insulin receptor substrates (IRSs) are a family of intracellular proteins that integrate and coordinate hormone, cytokine, and growth factor signaling. To date, four IRS proteins (IRS-1 to IRS-4) have been identified (27). All IRSs contain multiple tyrosine phosphorylation sites that act as binding sites for SH2-containing proteins (27). The IRS proteins were first identified as substrates and presumed signaling intermediates of the insulin receptor. However, it is now clear that the IRS proteins can be activated and phosphorylated by a number of other signaling pathways, including those that are critical for mammary gland development, such as growth hormone and prolactin (2, 56).Much research has focused on the roles of IRSs in both metabolic and mitogenic signaling; however, the last several years have seen an emergence of literature implicating IRSs in human cancer. IRS-1 is constitutively active and phosphorylated in many tumors (6). IRS-1 levels are increased in patients with pancreatic cancer (1), and both IRS-1 and IRS-2 levels are increased in patients with hepatocellular cancer (3, 36). We previously reported that high IRS-1 levels are associated with poor outcomes for patients with breast cancer (25, 41), and this is supported by further studies showing that IRS-1 is expressed in patients with primary breast cancer and metastases, and its levels correlate with poor differentiation and lymph node involvement (22). One study, however, found that IRS-1 levels in advanced primary breast cancers were reduced compared to breasts from healthy patients (44).The mouse mammary gland has served as a useful area for the identification and characterization of oncogenes and tumor suppressor genes important in human breast cancer (15). For example, transgenic mice overexpressing the HER-2 oncogene develop mammary cancer with biological and phenotypic variances similar to those observed in human breast cancer patients (23).To date, there have been no re...

Resistance of myeloma to lenalidomide is an emerging clinical problem, and though it has been associated in part with activation of Wnt/β-catenin signaling, the mediators of this phenotype remained undefined. Lenalidomide-resistant models were found to overexpress the hyaluronan (HA)-binding protein CD44, a downstream Wnt/β-catenin transcriptional target. Consistent with a role of CD44 in cell adhesion-mediated drug-resistance (CAM-DR), lenalidomide-resistant myeloma cells were more adhesive to bone marrow stroma and HA-coated plates. Blockade of CD44 with monoclonal antibodies, free HA, or CD44 knockdown reduced adhesion and sensitized to lenalidomide. Wnt/β-catenin suppression by FH535 enhanced the activity of lenalidomide, as did interleukin-6 neutralization with siltuximab. Notably, all-trans-retinoic acid (ATRA) down-regulated total β-catenin, cell-surface and total CD44, reduced adhesion of lenalidomide-resistant myeloma cells, and enhanced the activity of lenalidomide in a lenalidomide-resistant in vivo murine xenograft model. Finally, ATRA sensitized primary myeloma samples from patients that had relapsed and/or refractory disease after lenalidomide therapy to this immunomodulatory agent ex vivo. Taken together, our findings support the hypotheses that CD44 and CAM-DR contribute to lenalidomide-resistance in multiple myeloma, that CD44 should be evaluated as a putative biomarker of sensitivity to lenalidomide, and that ATRA or other approaches that target CD44 may overcome clinical lenalidomide resistance.

SUMMARY Proteasome inhibitors have revolutionized outcomes in multiple myeloma, but they are used empirically, and primary and secondary resistance are emerging problems. We have identified TJP1 as a determinant of plasma cell proteasome inhibitor susceptibility. TJP1 suppressed expression of the catalytically active immunoproteasome subunits LMP7 and LMP2, decreased proteasome activity, and enhanced proteasome inhibitor sensitivity in vitro and in vivo. This occurred through TJP1-mediated suppression of EGFR/JAK1/STAT3 signaling, which modulated LMP7 and LMP2 levels. In the clinic, high TJP1 expression in patient myeloma cells was associated with a significantly higher likelihood of responding to bortezomib and a longer response duration, supporting the use of TJP1 as a biomarker to identify patients most likely to benefit from proteasome inhibitors.

T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.

Background: Acquired proteasome inhibitor resistance emerges in myeloma patients through incompletely understood mechanisms. Results: Activation of nuclear factor (erythroid-derived 2)-like 2 (NRF2) and proteassemblin (POMP) was linked to bortezomib resistance, while their inhibition reversed resistance. Conclusion:The NRF2/POMP axis contributes to bortezomib resistance. Significance: NRF2/POMP axis inhibition can be translated to the clinic to reverse bortezomib resistance and induce chemosensitization.

The epidermal growth factor (EGF) and insulin-like growth factor (IGF) signaling pathways are critically involved in cancer development and progression. However, how these two signals cross-talk with each other to regulate cancer cell growth is not clearly understood. In this study, we found that EGF remarkably induced expression of major IGF signaling components, insulin receptor substrate (IRS)-1 and IRS-2, an effect that could be blocked by EGF receptor (EGFR) tyrosine kinase inhibitors. Although both extracellular signal-regulated kinase and c-Jun NH 2 -terminal kinase (JNK) signaling pathways were involved in the EGF up-regulation of IRS-1, the IRS-2 induction by EGF was specifically mediated by JNK signaling. Consistent with this, EGF increased IRS-2 promoter activity, which was associated with recruitment of activator protein-1 (AP-1) transcription factors and was inhibited by blocking AP-1 activity. Moreover, EGF treatment enhanced IGF-I and integrin engagement-elicited tyrosine phosphorylation of IRS and their downstream signaling, such as binding to phosphatidylinositol 3 ¶-kinase regulatory subunit p85. Finally, repressing the induction of IRS-2 levels abolished the EGF enhancement of cell motility, suggesting that increased IRS-2 is essential for the EGF regulation of breast cancer cell migration. Taken together, our results reveal a novel mechanism of cross-talk between the EGF and IGF signaling pathways, which could have implications in therapeutic applications of targeting EGFR in tumors. Because AP-1 activity is involved in breast cancer progression, our work may also suggest IRS-2 as a useful marker for aggressive breast cancer. (Cancer Res 2006; 66(10): 5304-13)

Mammary gland development is dependent upon the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis, this same axis has also been implicated in breast cancer progression. In this study we investigated the effect of a GH antagonist, pegvisomant (Somavert, Pfizer), on normal mammary gland development and breast cancer xenograft growth. Intraperitoneal administration of pegvisomant resulted in a 60% suppression of hepatic IGF-I mRNA levels and upto a 70-80% reduction of serum IGF-I levels. Pegvisomant administration to virgin female mice caused a significant delay of mammary ductal outgrowth that was associated with a decrease in the number of terminal end buds and reduced branching and complexity of the gland. This effect of pegvisomant was mediated by a complete inhibition of both GH and IGF-IR-mediated signaling within the gland. In breast cancer xenograft studies, pegvisomant caused shrinkage of MCF-7 xenografts, with an initial 30% reduction in tumor volume, which was associated with a 2-fold reduction in proliferation and a 2-fold induction of apoptosis. Long-term growth inhibition of MCF-7 xenografts was noted. In contrast, pegvisomant had no effect on MDA-231 or MDA-435 xenografts, consistent with primary growth of these xenografts being unresponsive to IGF-I both in vitro and in vivo. In MCF-7 xenografts that regressed, pegvisomant had only minor effects upon GHR and IGF-IR signaling. This data supports previous studies indicating a role for GH/IGF in mammary gland development, and suggests that pegvisomant maybe useful for the prevention and/or treatment of estrogen receptor positive breast cancer.

The c-Jun NH 2 -terminus kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here, we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphologic change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of activator protein transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2-mediated ERK activation, which in turn increased c-Fos expression and activator protein activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of extracellular signal-regulated kinase activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs. Mol Cancer Res; 8(2); 266-77. ©2010 AACR.