W. Gillies McKenna

Selective inhibition of Ras, phosphoinositide 3 kinase, and Akt isoforms increases the radiosensitivity of human carcinoma cell lines

Ras activation promotes the survival of tumor cells after DNA damage. To reverse this survival advantage, Ras signaling has been targeted for inhibition. Other contributors to Ras-mediated DNA damage survival have been identified using pharmacologic inhibition of signaling, but this approach is limited by the specificity of the inhibitors used and their toxicity. To better define components of Ras signaling that could be inhibited in a clinical setting, RNA interference was used to selectively block expression of specific isoforms of Ras, phosphoinositide 3 (PI3) kinase, and Akt. Inhibition of oncogenic Ras expression decreased both phospho-Akt and phospho-p42/44 mitogen-activated protein (MAP) kinase levels and reduced clonogenic survival. Because pharmacologic inhibition of PI3 kinases and Akt radiosensitized cell lines with active Ras signaling, whereas inhibition of the MAP/extracellular signal-regulated kinase (ERK) kinase/ERK pathway did not, we examined the contribution of PI3 kinases and Akts to radiation survival. Selective inhibition the PI3 kinase P110alpha + p85beta isoforms reduced Akt phosphorylation and radiation survival. Similarly, inhibition of Akt-1 reduced tumor cell radiation survival. Inhibition of Akt-2 or Akt-3 had less effect. Retroviral transduction and overexpression of mouse Akt-1 was shown to rescue cells from inhibition of endogenous human Akt-1 expression. This study shows that Ras signaling to the PI3 kinase-Akt pathway is an important contributor to survival, whether Ras activation results from mutation of ras or overexpression of epidermal growth factor receptor. This study further shows that selective inhibition of the PI3 kinase P110alpha + p85beta isoforms or Akt-1 could be a viable approach to sensitizing many tumor cells to cytotoxic therapies.

HIV Protease Inhibitors Block Akt Signaling and Radiosensitize Tumor Cells Both In vitro and In vivo

In tumor cells with mutations in epidermal growth factor receptor (SQ20B), H-Ras (T24), or K-Ras (MIAPACA2 and A549), the inhibition of Akt phosphorylation increases radiation sensitivity in clonogenic assays, suggesting that Akt is a potential molecular target when combined with therapeutic radiation. Insulin resistance and diabetes are recognized side effects of HIV protease inhibitors (HPIs), suggesting that these agents may inhibit Akt signaling. Because activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is common in human cancers, we hypothesized that HPIs can inhibit Akt activity resulting in increased tumor cell sensitivity to ionizing radiation-induced cell death. Five first-generation HPIs were subsequently tested and three of the five (amprenavir, nelfinavir, and saquinavir but not ritonavir or indinavir) inhibited Akt phosphorylation at Ser473 at serum concentrations routinely achieved in HIV patients. In both tumor cell colony formation assays and tumor regrowth delay experiments, combinations of drug and radiation exerted synergistic effects compared with either modality alone. In addition, in vivo, doses of amprenavir or nelfinavir comparable with the therapeutic levels achieved in HIV patients were sufficient to down-regulate phosphorylation of Akt in SQ20B and T24 xenografts. Finally, overexpression of active PI3K in cells without activation of Akt resulted in radiation resistance that could be inhibited with HPIs. Because there is abundant safety data on HPIs accumulated in thousands of HIV patients over the last 5 years, these agents are excellent candidates to be tested as radiation sensitizers in clinical trials.

Tumor Vascular Changes Mediated by Inhibition of Oncogenic Signaling

Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations of their vessels, including increased vascular maturity and acquisition of pericyte markers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Dual Inhibition of the PI3K/mTOR Pathway Increases Tumor Radiosensitivity by Normalizing Tumor Vasculature

The aberrant vascular architecture of solid tumors results in hypoxia that limits the efficacy of radiotherapy. Vascular normalization using antiangiogenic agents has been proposed as a means to improve radiation therapy by enhancing tumor oxygenation, but only short-lived effects for this strategy have been reported so far. Here, we show that NVP-BEZ235, a dual inhibitor of phosphoinositide-3-kinase (PI3K) and mTOR, can improve tumor oxygenation and vascular structure over a prolonged period that achieves the aim of effective vascular normalization. Because PI3K inhibition can radiosensitize tumor cells themselves, our experimental design explicitly distinguished effects on the blood vasculature versus tumor cells. Drug administration coincident with radiation enhanced the delay in tumor growth without changing tumor oxygenation, establishing that radiosensitization is a component of the response. However, the enhanced growth delay was substantially greater after induction of vascular normalization, meaning that this treatment enhanced the tumoral radioresponse. Importantly, changes in vascular morphology persisted throughout the entire course of the experiment. Our findings indicated that targeting the PI3K/mTOR pathway can modulate the tumor microenvironment to induce a prolonged normalization of blood vessels. The substantial therapeutic gain observed after combination of NVP-BEZ235 with irradiation has conceptual implications for cancer therapy and could be of broad translational importance.

COMBINED MODALITY TREATMENT OF ADULT SOFT TISSUE SARCOMAS OF THE HEAD AND NECK

Between 1975 and 1985, 16 adult patients high grade soft tissue sarcomas arising in the head and neck were treated with combined modality therapy at the National Cancer Institute. Fifteen patients underwent an attempt at gross resection, while only a biopsy was performed in 1 patient. All patients received conventionally fractionated, high-dose, post-operative irradiation. Twelve of 16 patients received adjuvant chemotherapy with cyclophosphamide and adriamycin, given concomitantly with radiation therapy. With a median follow-up of 43 months from diagnosis (range 11-116 months), 10 patients (64%) are disease-free. Of these 10 patients, 1 presented with pulmonary metastases and 2 patients later failed in the lung; all pulmonary disease was resected and these patients are disease-free at 80, 115, and 116 months following presentation. Local control was maintained in 12 patients (75%). An analysis of the iso-effect lines for local control shows a slope of 0.32. The median NSD for local control was 1720 ret. We conclude that aggressive local management of high grade sarcomas which combines gross resection and high-dose post-operative irradiation results in excellent local control. The role of adjuvant chemotherapy is less well defined in this small patient series. The acute and late complications of this combined modality approach are manageable.

RAS-mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines

Abstract Gupta, A. K., Bernhard, E. J., Bakanauskas, V. J., Wu, J., Muschel, R. J. and McKenna, W. G. RAS-Mediated Radiation Resistance is not Linked to MAP Kinase Activation. The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.

Modulation of the Tumor Microvasculature by Phosphoinositide-3 Kinase Inhibition Increases Doxorubicin Delivery In Vivo

Purpose: Because effective drug delivery is often limited by inadequate vasculature within the tumor, the ability to modulate the tumor microenvironment is one strategy that may achieve better drug distribution. We have previously shown that treatment of mice bearing tumors with phosphoinositide-3 kinase (PI3K) inhibitors alters vascular structure in a manner analogous to vascular normalization and results in increased perfusion of the tumor. On the basis of that result, we asked whether inhibition of PI3K would improve chemotherapy delivery. Experimental Design: Mice with xenografts using the cell line SQ20B bearing a hypoxia marker or MMTV-neu transgenic mice with spontaneous breast tumors were treated with the class I PI3K inhibitor GDC-0941. The tumor vasculature was evaluated by Doppler ultrasound, and histology. The delivery of doxorubicin was assessed using whole animal fluorescence, distribution on histologic sections, high-performance liquid chromatography on tumor lysates, and tumor growth delay. Results: Treatment with GDC-0941 led to approximately three-fold increases in perfusion, substantially reduced hypoxia and vascular normalization by histology. Significantly increased amounts of doxorubicin were delivered to the tumors correlating with synergistic tumor growth delay. The GDC-0941 itself had no effect on tumor growth. Conclusion: Inhibition of PI3K led to vascular normalization and improved delivery of a chemotherapeutic agent. This study highlights the importance of the microvascular effects of some novel oncogenic signaling inhibitors and the need to take those changes into account in the design of clinical trials many of which use combinations of chemotherapeutic agents. Clin Cancer Res; 18(1); 161–9. ©2011 AACR.

Is correction for lung density in radiotherapy treatment planning necessary

From 1978-981 a series of 30 patients with cancer of the esophagus were treated at the National Cancer Institute. Each of these patients had a CT scan of the chest taken in the treatment position, but prior to any treatment being given. Using these scans a retrospective analysis of the effect of lung density on delivered dose was performed. This indicated that failure to correct for tissue inhomogeneity results in a much higher dose being delivered than is prescribed. This effect is dependent on the energy of the beam being used for treatment; it may exceed 30% for 60Co. It also showed that there is wide patient to patient variation in lung density and that this variation is non-randomly distributed. The average lung density in his group of patients was 0.21 compared to the standard estimate of 0.35 but some had densities substantially lower than this, these being the patients with the largest lung volumes. This variability acts to further increase the discrepancy between prescribed and delivered dose even in a very homogeneous group of patients being treated under identical conditions for the same malignancy. The implications of this for future clinical trials in thoracic malignancies are discussed.

Gene expression profiling of hela cells in G1 or G2 phases

The cell division cycle is regulated through both transcriptional and post-transcriptional mechanisms. The altered expression of a number of genes at the mRNA level is known to be essential for progression through the cell cycle, however, a comprehensive gene expression profile of human cells remains to be completed. Here we sought to monitor the differential gene expression of genes after the transition of G2 cells into G1 prior to the restriction point. GeneChip containing microarrays of oligonucleotides corresponding to over 12 000 human genes were employed to profile differential gene expression in G1 and G2. After three independent experiments the resultant data was filtered and a set of genes was compiled based on at least threefold-altered expression, no background noise in determining expression and observation in all experiments. Our analysis identified 154 genes that were elevated in G2 phase of cells as compared to early G1 phase including 15 novel genes. This number included mRNAs whose upregulation is known to occur in G2 phase. Surprisingly only 19 genes were upregulated in G1 phase, among these six genes were novel. Some of these genes are candidates for transition through early G1. This gene inventory for G1 and G2 phases of cell cycle will provide the basis for understanding of cell cycle delay as a result of DNA damage.

Prognostic value, localization and correlation of PD-1/PD-L1, CD8 and FOXP3 with the desmoplastic stroma in pancreatic ductal adenocarcinoma

We examined the prognostic value of programmed cell death-1 (PD-1) and its ligand (PD-L1) together with CD8+ tumor-infiltrating lymphocytes (TILs) and FOXP3+ Tregs in resectable pancreatic ductal adenocarcinoma (PDAC) samples treated with adjuvant chemotherapy. Whole-mount FFPE tissue sections from 145 pancreatectomies were immunohistochemically stained for PD-1, PD-L1, CD8 and FOXP3. Their expression was correlated with clinicopathological characteristics, and overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), in the context of stroma density (haematoxylin-eosin) and activity (alpha-smooth muscle actin) and in regard to intratumoral lymphoid aggregates. The median OS was 21 months after a mean follow-up of 20 months (range, 2-69 months). In multivariate analysis, high PD-1+ TILs expression was associated with better OS (p = 0.049), LPFS (p = 0.017) and DMFS (p = 0.021). Similar findings were observed for CD8+ TILs, whereas FOXP3 and PD-L1 lacked prognostic significance. Although TIL distribution was heterogeneous, tumors of high stroma density had higher infiltration of CD8+ TILs than loose density stroma and vice versa (p < 0.001), whereas no correlation was found with stromal activity. Sixty (41.4%) tumors contained lymphoid aggregates and the presence of PD-1+ TILs was associated with better OS (p = 0.030), LPFS (p = 0.025) and DMFS (p = 0.033), whereas CD8+ TILs only correlated with superior LPFS (p = 0.039). PD-1+ and CD8+ TILs constitute independent prognostic markers in patients with PDAC treated with adjuvant chemotherapy. Our study provides important insight on the role of PD-1/PD-L1 in the context of desmoplastic stroma and could help guide future immunotherapies in PDAC.