Because the function of IGF1R in cancer biology has been well-documented, anti-IGF1R strategies are being tested clinically with antibodies and small molecule TKIs. While anti-IGF1R antibody therapies have advanced into Phase II and III clinical trials (Weroha and Haluska, 2008
), small molecule TKIs against both IGF1R and IR are just completing Phase I clinical trials. To date, the TKIs have not shown specificity for IGF1R; significant inhibition of IR has also been seen with these compounds (Haluska et al., 2006
; Ji et al., 2007
). This raises a very intriguing question: “Is IR a valuable target in cancer cells, even though systemic inhibition may result in metabolic toxicity?” This study provides direct evidence that downregulation of IR alone inhibits cancer cell proliferation, angiogenesis, lymphangiogenesis, and metastasis, suggesting that IR should also be targeted.
Using a human phosphor-kinase profiler array, we show that in LCC6 cells, insulin stimulation resulted only in the phosphorylation of Akt at Ser 473. In the IR shRNA clone cells, the phosphorylation of Akt at this site was diminished after insulin treatment. Although we cannot rule out the possibility that other signaling pathways may be different between LCC6 and the IR shRNA clones at other time points of insulin treatment, our data imply that Akt phosphorylation and activation play a critical role in the proliferative and metastatic phenotype of LCC6 cells. These results are consistent with recent findings in transgenic mouse model systems showing a role for Akt2 in pulmonary metastasis (Dillon et al., 2009
). As new PI3-K and Akt inhibitors are developed, the importance of insulin stimulation of this pathway in metastasis should be clarified (Maira et al., 2008
Downregulation of IR inhibited the anchorage independent growth of both LCC6 and T47D cells. Furthermore, downregulation of IR in LCC6 cells inhibited xenograft tumor growth in athymic mice. All of these data demonstrate the important function of the IR in proliferation. These data are not surprising because insulin has been shown to stimulate cancer cell proliferation for over three decades (Osborne et al., 1976
). Other evidence also supports the idea that breast tumor growth is insulin dependent and IR downregulation affects tumor growth (Heuson and Legros, 1972
; Novosyadlyy et al., 2009
). Insulin does not interact with IGF1R or the IGF1R/IR hybrids at physiological concentrations (Pandini et al., 1999
); thus, these effects are mainly mediated by IR. It is unknown whether one isoform or both isoforms of the insulin receptor play an important role in cell proliferation. The shRNA used in this study downregulated the level of both IR isoforms; thus, we are unable to attribute our results to a specific isoform. We and others have previously shown that IR-A, which binds insulin and IGF-II, is predominantly expressed in breast cancer specimens and breast cancer cell lines (Zhang et al., 2007
). Similarly, Avnet et al
. has shown that IR-A is expressed more than IR-B in human osteosarcoma samples (Avnet et al., 2009
). These data imply that IR-A may play a more important role in tumor biology. Future studies dissecting the differences between these two isoforms are needed to shed further light on the subject.
Furthermore, our data have shown that IR regulates angiogenesis and lymphangiogenesis of cancer cells. In the literature, VEGF-A produced by cancer cells plays a significant role in angiogenesis and the metastatic dissemination of cancer cells. Our results have shown that the insulin receptor regulates VEGF-A transcription and expression, which is consistent with literature that IGF-I and insulin regulate VEGF-A through hypoxia-inducible factor-1α (HIF1α) in other types of cancer (Feldser et al., 1999
; Fukuda et al., 2002
; Li et al., 2006
; Stoeltzing et al., 2003
; Treins et al., 2002
). Although a few reports have shown that IGF-I regulates lymphangiogenesis (Bjorndahl et al., 2005
; Tang et al., 2003
), we have failed to find literature regarding insulin and IR regulation of VEGF-D production and lymphangiogenesis in tumor cells. Therefore, our data suggest an important role of IR in lymphatic vessel formation in tumors.
Our data in LCC6 cells have shown that the IR, similar to IGF1R (Sachdev et al., 2004
), regulates metastasis in invasive cancer cells. Several molecular mechanisms mediated by IR may contribute to this phenotype. First, angiogenesis is essential to the metastatic dissemination of tumor cells to distant organs (Ademuyiwa and Miller, 2008
). Downregulation of IR in LCC6 cells significantly decreased the level of VEGF-A production, which may have contributed to both the reduced primary tumor proliferation at the mammary fat pad and the metastatic dissemination of tumor cells in the lung and liver of athymic mice. Second, lymphangiogenesis of IR shRNA xenograft tumors was reduced compared with wild type LCC6 xenografts. Lymphatic vessels are another pathway for tumor cells to disseminate (Eccles et al., 2007
). Third, through direct tail vein injection methods, we have shown that IR downregulation inhibits the colonization of LCC6 cells at distant sites including the lung and liver. This may be directly linked to the function of IR in cell survival and the ability to suppress anoikis. Finally, activation of IR signaling pathways might be required for colonization in specific host tissues. While the most relevant mechanism is not certain, it is clear that IR function is necessary for spontaneous metastases.
Our studies have explored the function of IR in cancer cell proliferation, angiogenesis, lymphangiogenesis and metastasis. Yet other aspects, such as tumor metabolism, have not been directly studied in these cells. Abnormalities in glucose metabolism (Warburg effect) is an increasingly “rediscovered” hallmark of cancer (Vander Heiden et al., 2009
). It has been shown that IR plays an important role in glucose metabolism of tumor cells (Frasca et al., 1999
). However, a substantial portion of glucose uptake is insulin-independent (Zhang et al., 2007
) and the direct effect of downregulation of IR on tumor metabolism needs further investigation.
Since both IGF1R and IR play important functions in cancer biology, TKIs, which do not distinguish between the two receptors, might be more beneficial to treat cancer patients if IR plays an important role in tumor cell biology. Because of the concern of diabetic side effects with IR inhibition, TKIs might be used on an intermittent schedule or in combination with other conventional or targeted therapies. Moreover, a role for IR needs to be considered in the conduct of ongoing clinical trials. We believe that measurement of both IGF1R and IR in tumors will be needed to interpret the results of clinical trials. While IGF1R is undoubtedly a target for cancer therapy, these data, along with those from other laboratories, suggest that IR may also be a target.