Thuy L. Phung

Phosphoinositide 3-Kinase Activity Is Necessary for Insulin-dependent Inhibition of Apolipoprotein B Secretion by Rat Hepatocytes and Localizes to the Endoplasmic Reticulum

Insulin inhibits apolipoprotein B (apoB) secretion by primary rat hepatocytes through activation of phosphoinositide 3-kinase (PI 3-K). Current studies demonstrate that the PI 3-K inhibitor wortmannin inhibits both basal and insulin-stimulated PI 3-K activities. Wortmannin and LY 294002, two structurally distinct PI 3-K inhibitors, prevent insulin-dependent inhibition of apoB secretion in a dose-dependent manner. To link PI 3-K activation to insulin action on apoB, we investigated whether insulin induced localization of activated PI 3-K to the endoplasmic reticulum (ER), where apoB biogenesis is initiated. Insulin action results in a significant redistribution of PI 3-K to a low density microsome (LDM) fraction containing apoB protein and apoB mRNA. Insulin stimulates a significant increase in PI 3-K activity associated with insulin receptor substrate-1 as well as an increase in insulin receptor substrate-1/PI 3-K mass in LDM. Subfractionation of LDM on sucrose density gradients shows that insulin significantly increases the amount of PI 3-K present in an ER fraction containing apoB. Insulin stimulates PI 3-K activity in smooth and rough microsomes isolated from rat hepatocytes, the latter of which contain rough ER as demonstrated by electron microscopy. Studies indicate that 1) PI 3-K activity is necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes; 2) insulin action leads to the activation and localization of PI 3-K in an ER fraction containing apoB; and 3) insulin stimulates PI 3-K activity in the rough ER.

Angiosarcoma: Clinical and molecular insights

Objective:Angiosarcoma (AS) is a rare understudied soft tissue sarcoma exhibiting endothelial cell differentiation. We sought to evaluate AS natural history in the largest patient cohort reported to date and further unravel commonly deregulated molecular events of potential therapeutic utility. Methods:Medical records of AS patients (n = 222) treated at our institution from 1993 to 2007 were reviewed. Univariable and multivariable analyses were used to identify independent outcome prognosticators. An AS tissue microarray (n = 68 human specimens) was constructed for immunohistochemical analysis of multiple potential drugable kinase-related molecular markers. Results:Forty-three (19.4%) metastatic AS patients and 179 patients (80.6%) with localized disease were included. Median survival of localized versus metastatic AS was 49 (range, 2–188) versus 10 (range, 1–69) months (P < 0.0001). Patients with localized AS who underwent complete surgical resection (n = 136; 76%) demonstrated significantly better outcome compared with those with unresectable tumors (n = 43; 24%; P < 0.0001). Of several factors identified on univariable analysis as significantly adverse for disease-specific survival, tumor size (>5 cm vs. ≤5 cm, P = 0.01) and epithelioid histologic component (P = 0.008) remained significant on multivariable analysis as independent adverse prognosticators in complete resection patients. Immunohistochemistry identified significant overexpression of vascular endothelial growth factor-A and C as well as p-AKT, p-4EBP1, and eIF4E in human AS. Conclusions:AS harbors a dismal outcome and even patients with disease amenable to complete surgical resection exhibit a 5-year disease-specific survival of only 53%. There is a crucial need for better therapies. Data presented here support further study of the AKT/mTOR pathway as novel molecular targets for AS therapy.

Rapamycin Inhibition of the Akt/mTOR Pathway Blocks Select Stages of VEGF-A164–Driven Angiogenesis, in Part by Blocking S6Kinase

Objective—We evaluated the stages of VEGF-A164 driven angiogenesis that are inhibited by therapeutic doses of rapamycin and the potential role of S6K1 in that response. Methods and Results—We assessed the effects of rapamycin on the several stages of angiogensis and lymphangiogenesis induced with an adenovirus expressing VEGF-A164 (Ad-VEGF-A164) in the ears of adult nude mice. Rapamycin (0.5 mg/kg/d) effectively inhibited mTOR and downstream S6K1 signaling and partially inhibited Akt signaling, likely through effects on TORC2. The earliest stages of angiogenesis, including mother vessel formation and increased vascular permeability, were strikingly inhibited by rapamycin, as was subsequent formation of daughter glomeruloid microvasular proliferations. However, later stage formation of vascular malformations and lymphangiogenesis were unaffected. Retrovirally delivered isoforms and shRNAs demonstrated that S6K1 signaling plays an important role in early VEGF-A164-angiogenesis. Conclusions—Rapamycin potently inhibited early and mid stages of VEGF-A164–driven angiogenesis, but not late-stage angiogenesis or lymphangiogenesis. Rapamycin decreased phosphorylation of both Akt and S6, suggesting that both the TORC1 and TORC2 pathways are impacted. Inhibition of S6K1 signaling downstream of mTOR is a major component of the antiangiogenesis action of rapamycin.

Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming

Blockade of angiogenesis can retard tumour growth, but may also paradoxically increase metastasis. This paradox may be resolved by vessel normalization, which involves increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability, and consequently mitigated hypoxia. Although these processes alter tumour progression, their regulation is poorly understood. Here we show that type 1 T helper (TH1) cells play a crucial role in vessel normalization. Bioinformatic analyses revealed that gene expression features related to vessel normalization correlate with immunostimulatory pathways, especially T lymphocyte infiltration or activity. To delineate the causal relationship, we used various mouse models with vessel normalization or T lymphocyte deficiencies. Although disruption of vessel normalization reduced T lymphocyte infiltration as expected, reciprocal depletion or inactivation of CD4+ T lymphocytes decreased vessel normalization, indicating a mutually regulatory loop. In addition, activation of CD4+ T lymphocytes by immune checkpoint blockade increased vessel normalization. TH1 cells that secrete interferon-γ are a major population of cells associated with vessel normalization. Patient-derived xenograft tumours growing in immunodeficient mice exhibited enhanced hypoxia compared to the original tumours in immunocompetent humans, and hypoxia was reduced by adoptive TH1 transfer. Our findings elucidate an unexpected role of TH1 cells in vasculature and immune reprogramming. TH1 cells may be a marker and a determinant of both immune checkpoint blockade and anti-angiogenesis efficacy.

Long-term blood vessel removal with combined laser and topical rapamycin antiangiogenic therapy: Implications for effective port wine stain treatment

Complete blanching of port wine stain (PWS) birthmarks after laser therapy is rarely achieved for most patients. We postulate that the low therapeutic efficacy or treatment failure is caused by regeneration and revascularization of photocoagulated blood vessels due to angiogenesis associated with the skins normal wound healing response. Rapamycin (RPM), an antiangiogenic agent, has been demonstrated to inhibit growth of pathological blood vessels. Our objectives were to (1) investigate whether topical RPM can inhibit reperfusion of photocoagulated blood vessels in an animal model and (2) determine the effective RPM concentration required to achieve this objective.

PHACE Syndrome: Current Knowledge, Future Directions

Abstract:  On November 7–8, 2008, physicians gathered in Houston Texas for the first‐ever workshop on PHACE syndrome, an important and recently described neurocutaneous syndrome. This article represents a summary of the discussions held at that workshop, which was attended by a broad range of medical specialists.

Endothelial Akt Signaling Is Rate-Limiting for Rapamycin Inhibition of Mouse Mammary Tumor Progression

Chronic activation of Akt signaling in the endothelium recapitulates the salient features of a tumor vasculature and can be inhibited by rapamycin, an inhibitor of mammalian target of rapamycin. This led to the hypothesis that the antitumor efficacy of rapamycin may be partially dependent on its ability to inhibit endothelial Akt signaling, making rapamycin an antiangiogenic agent and endothelial Akt pathway inhibitor. Dose-response studies with rapamycin showed that primary human endothelial cells and fibroblasts had a bimodal Akt response with effective reductions in phosphorylated Akt (pAkt) achieved at 10 ng/mL. In contrast, rapamycin increased pAkt levels in tumor cell lines. When tumor-bearing mice were treated with rapamycin doses comparable to those used clinically in transplant patients, we observed strong inhibition of mammary tumor growth. To test whether Akt activation in the endothelium was rate-limiting for this antitumor response, we engineered mouse mammary tumor virus-polyoma virus middle T antigen mice with endothelial cell-specific expression of constitutively activated Akt. We observed that the antitumor efficacy of rapamycin was reduced in the presence of elevated endothelial Akt activation. Just as we observed in MCF7 cells in vitro, rapamycin doses that were antiangiogenic resulted in increased pAkt levels in total mouse mammary tumor virus-polyoma virus middle T antigen tumor lysates, suggesting that tumor cells had an opposite Akt response following mammalian target of rapamycin inhibition compared with tumor endothelial cells. Together, these data support the hypothesis that endothelial Akt signaling in the tumor vasculature is an important target of the novel anticancer drug rapamycin.

Observations on Enhanced Port Wine Stain Blanching Induced by Combined Pulsed Dye Laser and Rapamycin Administration

Port wine stain (PWS) is a congenital, progressive vascular malformation of skin involving post-capillary venules that occurs in an estimated 3–4 children per 1,000 live births (1–3). Since most malformations occur on the face and neck, PWS is a clinically significant problem in the majority of patients. PWS are initially flat red macules that can be localized or segmental, but lesions tend to darken progressively, and by middle age, often transform into dark-red or purple plaques with nodularity (4, 5).

Accuracy of biopsy sampling for subtyping basal cell carcinoma.

BACKGROUND Basal cell carcinoma (BCC) is a common skin cancer for which the treatment and recurrence risk correlate with the histologic subtype. Limited information is available regarding the accuracy of biopsy in diagnosing BCC subtypes. OBJECTIVE We sought to determine the correlation between BCC subtypes present in a biopsy specimen and the actual subtypes present in a tumor. METHODS In this retrospective study, skin biopsy specimens and corresponding excisions were reviewed. All histologic subtypes present in the biopsy specimen were reported and compared with the composite BCC subtype present in the biopsy specimen and excision. RESULTS A total of 232 biopsy specimens and corresponding wide excisions were examined. The biopsy specimen accuracy rate was 82% for punch and shave biopsy specimens. Mixed histologic subtypes were seen in 54% of the cases, half of which contained an aggressive subtype (infiltrative, morpheaform, or micronodular). There was an 18% discordance rate between the biopsy specimen subtype and the composite subtype. Importantly, 40% of these discordant cases (7% of all cases examined) had an aggressive subtype that was not sampled in the initial biopsy specimen. Furthermore, some cases were misidentified as infiltrative subtype in the biopsy specimen as a result of misinterpretation of surface ulceration and reactive stromal changes. LIMITATIONS The limited number of punch biopsy specimens and the fact that Mohs excisions were not included are limitations. CONCLUSIONS Punch and shave biopsy specimens provided adequate sampling for correct BCC subtyping in 82% of the cases examined. However, 18% of the biopsy specimens were misidentified, some of which missed an aggressive component. Thus, there are potential pitfalls in the identification of BCC subtypes in biopsy specimens, which may have important implications in treatment outcome.

Regulation of hepatic apolipoprotein B RNA editing in the genetically obese Zucker rat

Hepatic apolipoprotein (apo) B RNA editing was examined in the genetically obese hyperinsulinemic and hypertriglyceridemic Zucker rat. In obese Zucker rats, apo B RNA editing was increased 42% relative to that in lean controls. Correspondingly, the proportion of serum triglyceride-rich lipoprotein containing apo B48 increased 4.7-fold in the obese Zucker rat. Quantification of hepatic total apo B mRNA showed no difference between obese Zucker and lean control rats. In contrast, the hepatic mRNA encoding APOBEC-1, the catalytic subunit of the RNA editing activity, demonstrated an increased abundance of 1.8-fold in obese Zucker rats versus lean controls.