Targeting AKT with the proapoptotic peptide, TAT-CTMP: a novel strategy for the treatment of human pancreatic adenocarcinoma

Abstract

Pancreatic adenocarcinoma carries an ominous prognosis and has little effective treatment. Several studies have demonstrated that the potently antiapoptotic phosphatidyl inositol 3'-kinase (PI3K)-protein kinase B/AKT pathway is active in pancreas cancer. A recent study identified an endogenous AKT antagonist, carboxyl terminal modulator protein (CTMP). CTMP inhibits the phosphorylation of AKT, preventing full activation of the kinase. We screened several cell permeable peptides from the N-terminal domain of CTMP (termed TAT-CTMP1-4) in vitro and found one that caused significant apoptosis in pancreatic adenocarcinoma cell lines. An inactive variant of this peptide was synthesized and used as a negative control. In all cell lines tested, TAT-CTMP4 induced a dose-dependent increase in apoptosis as detected by %-TUNEL positive cells and %-active caspase-3 (% active caspase-3 ranged from 31.2 to 61.9 at the highest dose tested (10 microM). A screening of various cell and tissue types revealed that the proapoptotic activity was highest in pancreatic adenocarcinoma. TAT-CTMP induced similar levels of active caspase-3 as several other known inducers of apoptosis: gemcitabine, radiation therapy, wortmannin and recombinant tumor necrosis factor (TNF)-alpha. No apoptosis was observed in donor human peripheral blood mononuclear cells (PBMC, p < 0.01). We further showed that TAT-CTMP4 could augment either gemcitabine chemotherapy or radiation therapy, standard therapies for pancreas cancer. Pancreatic adenocarcinoma xenografts treated with a single dose of TAT-CTMP4 demonstrated a marked increase in caspase-3 positive tumor cells when compared with untreated controls. Additionally, pancreatic adenocarcinoma allografts treated with intratumoral TAT-CTMP and systemic gemcitabine displayed a significantly smaller tumor burden while undergoing treatment than mice in control groups (p < 0.001). These data indicate that inhibiting AKT with CTMP may be of therapeutic benefit in the treatment of pancreatic adenocarcinoma and, when combined with established therapies, may result in an increase in tumor cell death.

Figure 1. Schematic structure of CTMP peptides and screening of the pro-apoptotic activity of CTMP-derived peptides in model pancreas adenocarcinoma

(A) Linear diagram of full-length CTMP. Areas outlined in red are predicted α-helices. Area outline in blue is a putative thioesterase domain. Cropped areas denote sequences from which CTMP peptides 1–4 were generated. (B) Helical wheel diagram of the active domain of CTMP4. The shaded residues are mutated from phenylalanine to alanine in the inactive control peptide (TAT-CTMP4 Inactive). (C) Human model pancreatic adenocarcinoma (Panc-1) were treated with TAT-CTMP peptides 1–4, and TAT-CTMP4-Inactive at a concentration of 10 µM. PBS alone and radiation therapy (2000 rads) were utilized as controls. After 18 hours of treatment, cells were harvested and % active caspase-3 was determined by flow cytometry. Each experimental group represents an n=3. Results are expressed as the mean, with bars representing standard error of the mean. *=P<0.01.

Figure 2. The induction of apoptosis by TAT-CTMP4 occurs via inhibition of the AKT pathway

(A) Model human pancreatic adenocarcinoma (CFPAC-1) were treated with escalating doses of TAT-CTMP4. Samples were treated for 1 hour prior to preparation of cell lysates. Western blots were subsequently prepared and stained with anti-total AKT and anti-phospho-AKT (Ser⁴⁷³). (B) Panc-1 lysates were generated after exposure to PBS (no treatment control), DMSO (vehicle control for wortmannin), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM) for 18 hours. Western blots were probed with anti-total GSK-3β and phospho-GSK-3α/β (Ser^21/9). (C) Human model pancreatic adenocarcinoma (CFPAC-1) were treated with PBS (no treatment), gemcitabine (30 nM), recombinant human TNF-α (10 ng/ml), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM). One hour later, cell lysates were generated and phospho-proteins were quantitated by bioplex analysis. Each experimental group represents an n=4. Results are expressed as the mean, with bars representing standard error of the mean.

Figure 3. Inhibition of the AKT pathway with the novel therapeutic, TAT-CTMP4, induces apoptosis in human and murine pancreatic adenocarcinoma in a dose-dependent fashion

(A) Representative FACS profile of model human pancreatic adenocarcinoma (Panc-1) treated with TAT-CTMP4 Inactive (10 µM) or TAT-CTMP4 (10 µM) and subsequently stained for active caspase-3. The human pancreatic adenocarcinoma cell line, Panc-1, was treated with PBS (no treatment), TAT-CTMP4-Inactive, and TAT-CTMP4 at 2.5, 5, and 10 µM concentrations for 18 hours. Cells were subsequently harvested and (B) % active caspase-3 and (C) % TUNEL positive cells were determined by flow cytometry. Each experimental group represents an n=3. (D) Assessment of % active caspase-3 in various model human (AsPC-1, BxPC-3, CFPAC-1) and a murine (Panc-02) pancreatic adenocarcinoma cell line treated with PBS (no treatment control), TAT-CTMP4 Inactive, and TAT-CTMP4 at a concentration of 10 µM for 18 hours. In all cases: * = P< 0.03. (E) Assessment of % active caspase-3 in various cell and tissue types. Samples were treated with PBS (no treatment control), TAT-CTMP4 Inactive, and TAT-CTMP4 at a concentration of 10 µM for 18 hours. Results are expressed as the mean, with bars representing standard error of the mean.

Figure 4. TAT-CTMP4 treatment does not induce apoptosis in human peripheral blood mononuclear cells (PBMC, A.), but does induce levels of apoptosis comparable to other known apoptosis-inducing agents (B.)

Human peripheral blood mononuclear cells were isolated from normal laboratory volunteers. 2.0 – 3.0 × 10⁶ PBMC were incubated for 18 h with PBS (no treatment control), TAT-CTMP4-Inactive (10 µM), TAT-CTMP4 (10 µM), or after being irradiated at 10,000 rads. % active caspase-3 was subsequently measured by flow cytometry. Each experimental group represents an n=5. Results are expressed as the mean, with bars representing standard error of the mean. * = p<0.01. (B) Human model pancreatic adenocarcinoma (Panc-1) were treated with PBS (no treatment), DMSO (vehicle control for wortmannin), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), TAT-CTMP4 (10 µM), recombinant human TNF-α (10 ng/ml), gemcitabine (30 nM), and radiation therapy (2000 rads). After 18 hours of treatment, cells were harvested and % active caspase-3 was determined by flow cytometry. Each experimental group represents an n=3. Results are expressed as the mean, with bars representing standard error of the mean.

Figure 5. Treatment of the chemo-resistant cell line, AsPC-1, utilizing combination therapy with TAT-CTMP4 in the presence of gemcitabine (A) and radiation therapy (B) results in an enhanced induction of apoptosis in model human pancreatic adenocarcinoma

The human pancreatic adenocarcinoma cell line, AsPC-1, was treated with PBS (no treatment), and submaximal doses of TAT-CTMP4 (2.5 and 5 µM), TAT-CTMP4-Inactive (2.5 and 5 µM) in the presence of gemcitabine (A, 30 nM) or radiation therapy (B, 2000 rads). After 18 hours of treatment, cells were harvested and % active caspase-3 was determined by flow cytometry. Results are expressed as the mean, with bars representing standard error of the mean.

Figure 6. Treatment of mice bearing heterotopic pancreatic adenocarcinoma xenografts with TAT-CTMP4 results in increased tumor apoptosis as detected by caspase-3 staining

The human pancreatic adenocarcinoma cell line, CFPAC-1, was implanted into the neck of nude mice (1 × 10⁶). After 10 days (mean tumor volume approx. 200 mm³), xenografts were treated with a single dose of intratumor PBS (200 µl), TAT-CTMP4 Inactive (400 µg), or TAT-CTMP4 (400 µg). Tumors were harvested 24 hours later and H&E and caspase-3 staining was subsequently performed on paraffin-embedded samples. Images presented were selected in a blinded fashion by an independent pathobiologist.

Figure 7. Treatment of mice bearing heterotopic pancreatic allografts with weekly gemcitabine and every other day TAT-CTMP results in inhibition of tumor growth compared to no treatment and gemcitabine-only controls

The murine pancreatic adenocarcinoma cell line, Panc-02, was implanted into the right flank of C57/BL6 mice (2.5 × 10⁵). After 14 days (mean tumor diameter approx 5 mm), treatment was initiated. Tumor bearing mice (n=10 per group) receiving gemcitabine were treated with intraperitoneal injections (1.5 mg/mouse, days 14 and 21). Mice receiving TAT-CTMP Inactive or TAT-CTMP were treated every other day for two weeks by intratumoral injection (0.5 mg/mouse, days 14, 16, 18, 20, 22, 24, 26). Results are expressed as the mean, with bars representing standard error of the mean.