Martha E. Lopez

p53 mutations are common in pancreatic cancer and are absent in chronic pancreatitis.

Pancreatic expression of the p53 tumor suppressor gene was studied in pancreatic adenocarcinomas and chronic pancreatitis. By immunohistochemistry, 16 of 34 (47%) cancers and none of the 24 chronic pancreatitis samples revealed nuclear staining. Sequence analysis indicated that 8 of 24 (33%) cancers were mutated for the p53 gene. Point substitutions occurred at codons 35, 105, 133, 213, 213, 258, and 299. A three base-pair in-frame insertion was identified between codons 261 and 262. None of 8 chronic pancreatitis samples exhibited p53 gene mutations. These data support a role for p53 gene alterations in human pancreatic cancer, and suggest that loss of its regulatory functions may constitute one of the differences between pancreatic cancer and chronic pancreatitis.

Attenuated ALK5 receptor expression in human pancreatic cancer: Correlation with resistance to growth inhibition

Transforming growth factor‐β (TGF‐β) receptors constitute a family of transmembrane proteins that bind TGF‐β ligands. In this study we assessed the growth responsiveness to TGF‐β1 in pancreatic cancer cell lines and characterized the levels of expression of TGF‐β receptors in these cell lines and in human pancreatic cancer tissues. COLO 357 cells were most sensitive to the growth inhibitory actions of TGF‐β1, PANC‐1 cells exhibited moderate sensitivity, Hs766T cells exhibited slight sensitivity and MIA PaCa‐2 and T3M4 cells were resistant to TGF‐β1. Only COLO 357 cells expressed high levels of ALK5, the major type I TGF‐β receptor (TβRI). Hs766T and PANC‐1 cells expressed high levels of SKRI, another TβRI subtype. Only MIA PaCa‐2 cells did not exhibit the type II TGF‐β receptor (Tβ‐RII) transcript, whereas type III TGF‐β receptor (Tβ‐RIII) mRNA levels were elevated in this cell line and in HS766T cells. All the cell lines expressed TGF‐β1, but TGF‐β2 and TGF‐β3 mRNA levels were variable. ALK5 and SKRI mRNA levels were 6.8‐ and 9‐fold greater in the pancreatic tumors in comparison with the corresponding levels in the normal pancreas. However, in the cancer cells, ALK5 immunoreactivity was faint, whereas TβRII immunoreactivity was focal and intense. Conversely, in ductal cells adjacent to cancer cells ALK5 immunoreactivity was strong, whereas TβRII immunoreactivity was weak. Since ALK5 heterodimerization with TβRII is crucial for TGF‐β‐mediated signaling, our findings suggest that low levels of ALK5 in pancreatic cancer cells within a tumor may protect against growth inhibition.

Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

Cells isolated from many types of human cancers express heparin-binding growth factors (HBGFs) that drive tumor growth, metastasis, and angiogenesis. The heparan sulfate proteoglycan glypican-1 (GPC1) is a coreceptor for HBGFs. Here we show that both cancer cell-derived and host-derived GPC1 are crucial for efficient growth, metastasis, and angiogenesis of human and mouse cancer cells. Thus downregulation of GPC1 in the human pancreatic cancer cell line PANC-1, using antisense approaches, resulted in prolonged doubling times and decreased anchorage-independent growth in vitro as well as attenuated tumor growth, angiogenesis, and metastasis when these cells were transplanted into athymic mice. Moreover, athymic mice that lacked GPC1 exhibited decreased tumor angiogenesis and metastasis following intrapancreatic implantation with either PANC-1 or T3M4 human pancreatic cancer cells and fewer pulmonary metastases following intravenous injection of murine B16-F10 melanoma cells. In addition, hepatic endothelial cells isolated from these mice exhibited an attenuated mitogenic response to VEGF-A. These data indicate that cancer cell- and host-derived GPC1 are crucial for full mitogenic, angiogenic, and metastatic potential of cancer cells. Thus targeting GPC1 might provide new avenues for cancer therapy and for the prevention of cancer metastasis.

Suppression of fibroblast growth factor receptor signaling inhibits pancreatic cancer growth in vitro and in vivo

BACKGROUND & AIMS Fibroblast growth factors (FGFs) are mitogenic polypeptides that activate specific cell surface FGF receptors (FGFRs). Pancreatic cancers overexpress basic FGF (bFGF) and the type I FGF receptor (FGFR-1), and overexpression of bFGF has been correlated with decreased patient survival. The aim of this study was to examine the effects of abrogation of FGFR-1-dependent signaling on pancreatic cancer cell growth. METHODS PANC-1 human pancreatic cancer cells were transfected with a truncated FGFR-1 complementary DNA (FGFR405), resulting in the expression of a kinase-deficient receptor. Activation of endogenous FGFR-1 was assessed in immunoblot studies with antiphosphotyrosine and anti-active mitogen-activated protein (MAP) kinase antibodies. Effects on cell growth were determined in vitro and in nude mice. RESULTS PANC-1 clones expressing the truncated receptor showed attenuated receptor tyrosine phosphorylation and MAP kinase activation in response to bFGF, decreased basal cell growth, and a marked decrease in tumor-forming potential in vivo. Confirmatory experiments with MIA PaCa-2 pancreatic cancer cells indicated that FGFR405 also attenuated FGF-dependent MAP kinase activation in this cell line. CONCLUSIONS The findings suggest that FGFR-dependent signaling is crucial for pancreatic cancer growth and raise the possibility that inhibition of FGFR signaling may ultimately prove useful as a therapeutic option in patients with pancreatic cancer.

Transfection of the type I TGF-β receptor restores TGF-β responsiveness in pancreatic cancer

Transforming growth factor‐beta (TGF‐β) signaling is initiated following heterodimerization of the type II TGF‐β receptor (TβRII) with the type I TGF‐β receptor (TβRI). Both receptors are required for TGF‐β responsiveness. In the present study, we characterized the actions of TGF‐β1 in T3M4 human pancreatic cancer cells, which express low levels of TβRI and high levels of TβRII. Cells were transiently transfected with p3TP‐Lux, a TGF‐β‐responsive luciferase reporter gene construct. TGF‐β1 was without effect in parental T3M4 cells, but caused a time‐ and dose‐dependent increase in luciferase activity in T3M4 cells co‐transfected with a TβRI cDNA expression vector. Co‐transfection of TβRI with a truncated Smad4 cDNA that is known to block TGF‐β‐dependent signaling, abrogated the TβRI‐induced increase in luciferase activity. Sequencing of the TβRI and the Smad4 genes in T3M4 cells did not reveal any mutations. These findings indicate that one mechanism for TGF‐β resistance in pancreatic cancer is due to a quantitative decrease in TβRI expression. Int. J. Cancer 78:255–260, 1998.© 1998 Wiley‐Liss, Inc.

Expression of a truncated EGF receptor is associated with inhibition of pancreatic cancer cell growth and enhanced sensitivity to cisplatinum

Human pancreatic cancers over‐express the epidermal growth factor receptor (EGF‐R) and all 5 known ligands of the EGF family, including EGF, transforming growth factor‐alpha (TGF‐α), amphiregulin, betacellulin and heparin‐binding EGF‐like growth factor (HB‐EGF). The aim of the present study was to confirm the presence of EGF‐R‐dependent autocrine loops in a human pancreatic cancer cell line and to explore the possibility that interrupting EGF‐R activation by introducing a truncated receptor abrogates pancreatic cancer cell growth. The anchorage‐independent growth of PANC‐1 human pancreatic cancer cells, previously shown to express TGF‐α, was inhibited by specific anti TGF‐α antibodies. PANC‐ 1 cells were then either transfected with an expression plasmid encoding a kinase‐deficient EGF‐R cDNA (HER653) or infected with the same EGF‐R cDNA using a retroviral vector. Multiple transfected and infected clones co‐expressed the truncated EGF‐R and endogenous EGF‐R as revealed by Northern blot analysis and immunoblots. In these clones, there was a marked attenuation in EGF‐ and TGF‐α‐mediated EGF‐R tyrosine phosphorylation and c‐tos induction. There was also a significant decrease in colony formation in soft agar by comparison with control cells and a significant increase in the effect of the growth‐inhibitory effect of the alkylating agent cisplatinum in these clones. Our observations indicate that dominant negative inhibition of EGF‐R may have therapeutic potential in pancreatic cancer.

Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors

Pancreatic ductal adenocarcinomas (PDACs) overexpress various cell-surface tyrosine kinase receptors, including the type I high-affinity fibroblast growth factor receptor (FGFR-1). The purpose of this study was to determine whether FGFR-targeted gene therapy is feasible in this disorder. Accordingly, the effects of a conjugate consisting of fibroblast growth factor (FGF)-2 linked to a Fab′ fragment against the adenovirus knob region were evaluated in human pancreatic cancer cell lines treated with an adenoviral vector containing the herpes simplex virus thymidine kinase (AdTK) gene. An adenoviral vector containing the firefly luciferase reporter gene (AdLuc) served to assess infection efficiency, and was initially tested in L6 rat myoblasts. In parental L6 cells that express exceedingly low levels of high-affinity FGFRs, transduction with AdLuc was enhanced 7- to 10-fold with the FGF2–Fab′ conjugate, whereas in L6 cells transfected to express FGFR-1, it was enhanced 39- to 52-fold. The pancreatic cancer cell lines expressed variable levels of the four high-affinity FGF receptors, and exhibited 2- to 34-fold increases in gene transduction in the presence of the FGF2–Fab′ conjugate. In the absence of FGF2–Fab′ there was no correlation between surface binding of FGF2 and AdLuc transduction efficiency, whereas in the presence of FGF2–Fab′, enhanced AdLuc transduction efficiency correlated with greater surface binding of FGF2. In the absence of AdTK, all the cell lines were insensitive to ganciclovir, whereas after AdTK transduction, only ASPC-1 and PANC-1 cells were resistant to ganciclovir even in the presence of FGF2–Fab′. Ganciclovir-mediated inhibition was dependent on the conjugate in CAPAN-1 and COLO-357 cells, but was independent of the conjugate in T3M4 and MIA-PaCa-2 cells. Real-time quantitative PCR of laser-captured cancer cells revealed high levels of various FGFR mRNA species in six of seven PDAC tumor samples. These findings indicate that transduction efficiency with FGF2–Fab′ in pancreatic cancer cells is independent of native adenoviral transduction efficiency and is greatest in cells that exhibit concomitant expression of various high-affinity FGFRs. In view of the overexpression of high-affinity FGFRs in the cancer cells in PDAC, our findings also suggest that the combined use of AdTK, ganciclovir, and FGF2–Fab′ may ultimately be a promising therapeutic approach in a subgroup of patients with PDAC.

Altered expression of insulin-like growth factor II receptor in human pancreatic cancer

The insulin-like growth factor-II (IGF-II) receptor (IGF-IIR) is a single-chain transmembrane protein identical to the mannose-6-phosphate receptor. In the present study we examined IGF-IIR expression in normal and cancerous human pancreatic tissues. In the normal pancreas, moderately strong IGF-IIR immunoreactivity was present in the cytoplasm of islet cells, and mild cytoplasmic immunoreactivity was evident occasionally in ductal and acinar cells. Some ductal cells also exhibited nuclear IGF-IIR immunoreactivity. In the pancreatic cancers, regions of strong IGF-IIR immunoreactivity were present in the duct-like cancer cells within the tumor mass, often exhibiting nuclear localization. Expression of IGF-IIR mRNA in the cancer cells was confirmed by in situ hybridization. By comparison with normal pancreatic tissues, 7 of 12 pancreatic cancers exhibited a 5.6-fold increase in IGF-IIR mRNA levels, whereas in 3 cancers the IGF-IIR transcript was below the level of detection. Furthermore, all six tested cultured human pancreatic cancer cell lines expressed the IGF-IIR mRNA transcript. Our data indicate that IGF-IIR is over expressed in a significant number of human pancreatic cancers, where it has a tendency to localize in the nucleus, and raise the possibility that IGF-IIR may contribute to the pathobiology of pancreatic cancer.

A Novel Type I Fibroblast Growth Factor Receptor Activates Mitogenic Signaling in the Absence of Detectable Tyrosine Phosphorylation of FRS2

A novel variant of the fibroblast growth factor receptor type 1 (FGFR-1) was identified in human placental RNA. In this receptor (FGFR-1L) portions of the second and third immunoglobulin-like (Ig-like) domains are deleted. To determine whether FGFR-1L was functional, full-length variant (pSV/FGFR-1L) and wild-type (pSV/FGFR-1) receptors were stably transfected into rat L6 myoblasts cells. Transfected L6 clones expressed respective proteins and bound125I-labeled FGF-2 withK d values of 99 pm (FGFR-1) and 26 pm (FGFR-1L). FGF-1 and FGF-2 competed efficiently with 125I-FGF-2 for binding to FGFR-1 and FGFR-1L, whereas FGF-4 was less efficient. FGF-1, FGF-2, and FGF-4 enhanced mitogen-activated protein kinase (MAPK) activity, increased steady-state c-fos mRNA levels, and stimulated proliferation through either receptor, whereas KGF was without effect. FGFR-1 expressing clones exhibited ligand-induced tyrosine phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2), a 90-kDa adaptor protein that links FGFR-1 activation to the MAPK cascade. In contrast, tyrosine phosphorylation of FRS2 was not evident with FGFR-1L. In addition, phospholipase C-γ was not tyrosine phosphorylated via activated FGFR-1L. These findings indicate that FGFR-1L binds FGF-1 and FGF-2 with high affinity and is capable of mitogenic signaling, but may activate MAPK to occur via non-classical signaling intermediates.

Expression of the IIIc Variant of FGF Receptor-1 Confers Mitogenic Responsiveness to Heparin and FGF-5 in TAKA-1 Pancreatic Ductal Cells.

SummaryBackground. Fibroblast growth factors (FGFs) contribute to angiogenesis and mitogenesis by binding to tyrosine kinase receptors termed FGF receptors (FGFRs). FGF-5 is a secreted FGF that is believed to preferentially act via the IIIc splice variant of FGFR-1. Human pancreatic ductal carcinoma cells express FGF-5 and FGFR-1 IIIc, implying a potential for autocrine growth modulation.Aim. In this study we investigated the importance of FGFR-1 IIIc expression for FGF-5 mitogenic signaling in a pancreatic ductal cell line.Methods. A cDNA encoding FGFR-1 IIIc was expressed in the well-differentiated TAKA-1 Syrian hamster pancreatic ductal cell line.Results. TAKA-1 cells secrete FGF-5, but were found not to express FGFR-1 and to be unresponsive to exogenous FGF-5. In contrast, TAKA-1 clones expressing FGFR-1 IIIc were growth stimulated in the presence of FGF-5 and displayed enhanced mitogen-activated protein kinase (MAPK) activity in the presence of FGF-5. PD98059, an inhibitor of this pathway, inhibited FGF-5-induced growth in these clones.Conclusion. Our data demonstrate that FGFR-1 IIIc can mediate FGF-5-induced mitogenesis via the MAPK pathway in pancreatic ductal cells, and suggest that expression of FGFR-1 IIIc in conjunction with FGF-5 may contribute to the pathobiology of human pancreatic cancer.