Brent W. Sutherland

Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors.

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.

Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells

Akt/PKB is a serine/threonine kinase that promotes tumor cell growth by phosphorylating transcription factors and cell cycle proteins. There is particular interest in finding tumor-specific substrates for Akt to understand how this protein functions in cancer and to provide new avenues for therapeutic targeting. Our laboratory sought to identify novel Akt substrates that are expressed in breast cancer. In this study, we determined that activated Akt is positively correlated with the protein expression of the transcription/translation factor Y-box binding protein-1 (YB-1) in primary breast cancer by screening tumor tissue microarrays. We therefore questioned whether Akt and YB-1 might be functionally linked. Herein, we illustrate that activated Akt binds to and phosphorylates the YB-1 cold shock domain at Ser102. We then addressed the functional significance of disrupting Ser102 by mutating it to Ala102. Following the stable expression of Flag:YB-1 and Flag:YB-1 (Ala102) in MCF-7 cells, we observed that disruption of the Akt phosphorylation site on YB-1 suppressed tumor cell growth in soft agar and in monolayer. This correlated with an inhibition of nuclear translocation by the YB-1(Ala102) mutant. In conclusion, YB-1 is a new Akt substrate and disruption of this specific site inhibits tumor cell growth.

Conditional Deletion of Rb Causes Early Stage Prostate Cancer

Prostate cancer remains the second leading cause of cancer-related death for men in the United States. Mutations in tumor suppressor genes including retinoblastoma (Rb), p53, and PTEN have been linked to the development of prostate cancer in man and mouse models, and loss of heterozygosity of the Rb locus has been observed in up to 60% of clinical cases. In this study we demonstrate that conditional somatic deletion of even a single Rb allele in the epithelial cells of the mouse prostate causes focal hyperplasia, thereby establishing a causal relationship between Rb loss and development of early stage prostate cancer. As a consequence of Rb ablation we observed increased expression of E2F target genes and a concomitant increase in proliferation in the epithelial compartment. However, by 52 weeks of age these lesions had not become malignant and represent an early stage of the disease. Nevertheless, the multifocal nature of the phenotype in the mice closely resembled multifocality of clinical disease. Taken together, our data demonstrated that loss of pRB-mediated cell cycle control directly caused the initiation of proliferative prostate disease but was insufficient to cause malignancy. Establishment of this early initiation model will aid efforts to thoroughly characterize early prostate disease as well as the elucidation of molecular mechanisms that cooperate with Rb loss to facilitate progression and metastasis.

Vasculature‐targeted tumor necrosis factor‐alpha increases the therapeutic index of doxorubicin against prostate cancer

Poor penetration and uneven distribution of doxorubicin in tumors limits the efficacy of this drug in patients with prostate cancer (PC). Aim of the study was to investigate whether pre‐treatment with NGR‐TNF, a tumor necrosis factor‐α derivative able to target tumor vessels and alter vessel permeability, increases the penetration and the efficacy of doxorubicin in pre‐clinical models of PC.

The involvement of insulin-like growth factor 2 binding protein 3 (IMP3) in pancreatic cancer cell migration, invasion, and adhesion

BackgroundOver-expression of insulin-like growth factor 2 mRNA binding protein 3 (IMP3) is correlated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Previous studies examining other cancer types have implicated IMP3 in the regulation of several cellular functions that are characteristic of tumour cells. However, the role of this oncofetal protein in PDAC progression remained unclear.MethodsUsing siRNA, we examined the effect of IMP3 inhibition on the motility, invasive ability, and matrix adhesion of PDAC cells. In addition, we also evaluated the expression of cytoskeleton-associated genes following IMP depletion.ResultsKnockdown of IMP3 significantly decreased the motility, invasion, and extracellular matrix adhesion of select PDAC cells in vitro. In addition, IMP3-depleted cells exhibited lower levels of CD44 protein and KIF11 mRNA. Moreover, we also observed a reduction in downstream RhoA signaling following IMP3 knockdown, indicating that IMP3 modulates the levels of proteins involved in cytoskeletal organization.ConclusionsThese results suggest that IMP3 facilitates PDAC progression by enhancing the pro-metastatic behaviour of tumour cells.

Lipid-based nanoformulation of irinotecan: dual mechanism of action allows for combination chemo/angiogenic therapy

A number of studies have outlined the antiangiogenic effects of cytotoxic agents when administered frequently at low doses. These studies suggest that the effect of the cytotoxic agent is on the vasculature within the tumor and it is assumed that there is little or negligible cytotoxicity. Liposomal drug delivery systems have the ability to provide a dual mechanism of activity where tumor accumulation can deliver high local concentrations of the drug at the site of action with concomitant slow release of the drug from carriers in the blood compartment that results in antivascular effects, similar to that achieved when dosing frequently at low levels. Although this dual mechanism of activity may be linked to other lipid nanoparticle formulations of anticancer drugs, this article summarizes the evidence supporting direct (cytotoxic) and indirect (antivascular) actions of a liposomal formulation of irinotecan.

Abstract LB-41: Regulation of adhesion, motility, and invasion by insulin-like growth factor-2 binding protein 3 in pancreatic cancer cells

Background: Overexpression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3 or IMP3), a member of the VICKZ family of RNA-binding proteins, has been correlated with poor prognosis in pancreatic cancer and other cancer types, including lung, bladder, kidney, liver, and prostate. In human leukemia cells as well as in HeLa and hepatocellular carcinoma cells, IMP3 has previously been shown to regulate a number of cellular functions. However, the role of this oncofetal protein in pancreatic cancer progression remains to be elucidated. In the present study, we characterized the effect of IMP3 inhibition on the proliferation, motility, adhesion, and invasive potential of pancreatic cancer cells. Methods: IMP3 levels in Panc-1 and Hs766T were decreased through RNA interference. Untreated and scramble-siRNA transfected cells were used as controls. The effect of knockdown on cellular behavior was evaluated 48 h after transfection. Proliferation was assessed using the MTS assay, while a commercially-available colorimetric assay was used to assess adhesion to extracellular matrix proteins. Motility and invasive potential were evaluated using the Boyden chamber assay. Data were analyzed using repeated measures ANOVA. Significant F ratios were further examined using the Tukey post-hoc test. Results: The basal levels of IMP3 were found to be similar in Panc1 and Hs766T cells. IMP3 siRNA knockdown reduced IMP3 protein levels by 48% relative to scramble siRNA control. In both cell lines, a decrease in IMP3 did not significantly affect proliferation when compared to scramble siRNA-treated control. Interestingly, knockdown of IMP3 reduced the adhesion of Hs766T to fibronectin (20%; P Conclusion: Our preliminary results demonstrated that IMP3 differentially regulates cellular adhesion and invasive potential of pancreatic cancer cell lines, suggesting that this oncofetal protein may play a role in tumor metastasis. In line with this hypothesis, we are currently examining the role of IMP3 in regulating the phenotype of L3.6pl, a pancreatic cancer cell line characterized to exhibit metastatic potential. In addition, studies are currently underway to identify the downstream molecular target(s) of IMP3 that may mediate metastasis of pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-41. doi:10.1158/1538-7445.AM2011-LB-41