Previous studies have identified three distinct stages of T-antigen-induced beta-cell tumor progression including islet hyperplasia, islet-angiogenesis and finally formation of solid encapsulated tumors 
. The predominant determinant in the progression of normal islets to a stage of hyperplasia and angiogenesis was found to be the rate of beta-cell proliferation, while reduced apoptosis was critical in the progression to solid encapsulated tumors 
. As expected, we observed a similar progression to insulinomas and associated hyperinsulinemia-hypoglycemia in wildtype-βTag
mice. However, beta-cell proliferation was significantly reduced in Perk-
mice and this defect was most prominent in the initial transition to islet hyperplasia. Insulinomas in Perk KO-βTag
mice were fewer, the average size was 38-fold smaller, and they rarely develop into encapsulated tumors. Cell death was slightly higher in wildtype-βTag
mice and therefore does not appear to contribute to the smaller size of tumors in Perk KO-βTag
mice. Beta-cells isolated from Perk KO-βTag
insulinomas also showed poor growth characteristics in culture, and we were unable to establish a T-antigen, Perk
-deficient beta-cell line from isolated insulinomas from these mice whereas we could readily establish them from the wildtype-βTag
mice. Although the T-antigen induction of beta-cell proliferation in Perk KO
was blunted compared to the wild-type mice, the increase in beta-cell mass was nonetheless sufficient to eventually reverse the diabetes of these mice.
During the later stages of tumor development hypoxia becomes more prevalent as cell growth outstrips the pre-existing vascular system 
. To provide increased circulation within the tumor microenvironment, angiogenesis is induced in part by the response to hypoxia 
. PERK has been shown to be important for regulating the hypoxic stress response in fibroblast-derived tumors 
and it was speculated that the slow growth of Perk
-deficient tumors was caused by a defect in the hypoxia-stress response resulting in increased cell death. We found that Perk
-deficient insulinomas failed to develop extensive vasculature, consistent with the hypothesis that PERK plays a critical role in regulating the response to hypoxia. However, we discovered that beta-cell proliferation and beta-cell mass in Perk KO-βTag
islets substantially lagged behind wildtype-βTag
islets during the initial hyperplasia stage prior to the time that hypoxic conditions exist. Moreover, beta-cell death was actually slightly higher in the islets of the wildtype-βTag
mice during the exponential growth phase and therefore does not contribute to the relatively small size of the Perk KO-βTag
islets. Thus we suggest that PERK has two roles in tumor formation, first to support rapid cell proliferation and then later to promote angiogenesis. In the absence of PERK, however, tumors may not achieve sufficient size in order for severe hypoxia and cell death to occur. We found high apparent beta-cell death only in a very large wildtype-βTag
insulinoma, but similarly sized insulinomas were never seen in Perk KO-βTag
mice. To examine PERK's role in late stage tumor progression it will be necessary to acutely ablate PERK expression later in tumor development.
In addition to regulating beta-cell functions that contribute tumor progression, PERK may also regulate functions in other cell types including the vascular endothelial cells that comprise the tumor macroenvironment. In our studies Perk is deficient in all cells of the entire animal, and therefore we cannot discriminate between PERK's functions in these different cell types. We are currently generating Tag-induced insulinomas in mice in which Perk has been specifically ablated in various cell types of the pancreas to determine the role of PERK in both the beta-cells and macroenvironment of the developing tumor.
The molecular mechanism underlying PERK-dependent regulation of tumor growth and angiogenesis is unknown. We speculate that reduced proliferation and vascularity in Perk
-deficient insulinomas is caused by a fundamental defect in function in the secretory pathway that we have recently discovered in beta-cells. Perk
-deficient beta-cells have defects in 
ER to Golgi trafficking, ER associated proteasomal degradation, and integrity of the ER and Golgi (unpublished). Cell proliferation and membrane targeting of angiogenic receptors (e.g. VEGFRs) are highly dependent upon normal ER functions. We therefore suggest that these defects in ER functions may negatively impact beta-cell proliferation and angiogenesis.
All the procedures that involved animal subjects were approved by the IACUC of the Pennsylvania State University in accordance with federal and state regulations governing care and use of animals.