A. Raffaele Bianco

Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy

Purpose: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase. Experimental Design: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student’s t test. All Ps were two-sided. Results: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80–90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor α of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5–10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474. Conclusions: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.

The Prognostic Value of Lymphatic and Blood Vessel Invasion in Operable Breast Cancer

Background. This study assessed the prognostic effect of lymphatic and blood vessel invasion (LVI and BVI) on survival in a retrospective sample of 1408 patients with breast cancer.

The RIα subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-Receptor

Functional interactions between protein kinase A (PKA) and epidermal growth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or its regulatory subunit RIα are generally overexpressed in cancer cells and are induced following transforming growth factor α (TGFα)/EGF-R-dependent transformation. Downregulation of RIα/PKAI inhibits TGFα expression and EGF-R-dependent signalling. We have previously shown that addition of EGF to quiescent human normal epithelial MCF-10A cells determines PKAI expression and cell membrane translocation before cells enter S phase, while PKAI inhibition prevents S phase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstrate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A cells. Moreover, induction of mitogen-activated protein kinase (MAPK) activity following EGF-R activation is mimicked by PKAI overexpression and inhibited by downregulators of PKAI. Finally, the PKAI – EGF-R association occurs through the binding of RIα to the SH3 domain(s) of Grb2 adaptor protein, thus allowing the recruitment of the PKAI holoenzyme to the activated EGF-R. This is the first demonstration of a direct interaction of PKAI with the activated EGF-R macromolecular signalling complex.

Differential immunohistochemical detection of transforming growth factor α, amphiregulin and CRIPTO in human normal and malignant breast tissues

The expression of growth factors, such as transforming growth factor α (TGFα), amphiregulin (AR) and CRIPTO, a type‐1 tyrosine‐kinase growth factor receptor‐(erbB‐2), and a tumor‐suppressor gene (p53), that have been implicated in the development and/or the progression of breast cancer, was evaluated by immunohistochemistry in 100 human primary infiltrating breast carcinomas (IBC). AR and CRIPTO immunoreactivity was also assessed in 55 human breast ductal carcinomas in situ (DCIS). Within the 100 IBC, 80, 50, 73, 17, and 34 tumors expressed moderate to high levels of TGFα, AR, CRIPTO, erbB‐2, and p53 respectively. In addition, AR and CRIPTO immunoreactivity were found in 11 and in 26 out of 55 DCIS respectively. In contrast, only 4, 3, and 2 out of 10 normal mammary‐gland samples were weakly positive for TGFα, AR, and CRIPTO expression, respectively, whereas none was positive for erbB‐2 or p53. Within the 100 IBC, expression of erbB‐2 significantly correlated with high histologic and nuclear grading, with high growth fraction, and with estrogen‐receptor(ER)‐ and progesterone‐receptor(PgR)‐negative tumors. A statistically significant correlation was also observed between p53 expression and high histologic grading, high growth fraction, and PgR‐negative tumors. In contrast, no significant correlations were found between TGFα, AR, and CRIPTO immunoreactivity and various clinicopathological parameters, with the exception of a positive correlation between TGFα and ER expression. These data demonstrate that TGFα, AR, and CRIPTO expression are significantly increased in malignant mammary epithelium relative to normal epithelium. In particular, the differential expression of CRIPTO may serve as a potential tumor marker for breast carcinogenesis.

ZD1839 (IRESSA), an EGFR-selective tyrosine kinase inhibitor, enhances taxane activity in bcl-2 overexpressing, multidrug-resistant MCF-7 ADR human breast cancer cells.

Constitutive bcl‐2 overexpression increases the tumorigenic and metastatic potential of doxorubicin‐resistant, estrogen‐independent, MCF‐7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl‐2‐overexpressing MCF‐7 ADR clones and control neomycin‐transfected MCF‐7 ADR neo cells. The 2 bcl‐2‐overexpressing MCF‐7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF‐7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF‐7 ADR neo and bcl‐2‐overexpressing MCF‐7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor‐α (TGF‐α). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa™, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR‐TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose‐dependent manner (IC50 of approximately 0.1 μM). This effect was accompanied by a dose‐dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF‐α, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl‐2‐overexpressing MCF‐7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose‐dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl‐2 expression in bcl‐2‐overexpressing MCF‐7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone‐independent, multidrug‐resistant, human breast cancer.

Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis and cooperate with cytotoxic drugs in human cancer cell lines

We have constructed a series of 22 phosphorothioate 20‐mer antisense oligonucleotides directed against different regions of the human (EGFR) mRNA. Treatment with EGFR antisense oligonucleotides showed a dose‐dependent inhibition of human GEO colon cancer cell growth in soft agar. Western blot analysis demonstrated a significant reduction in EGFR expression after treatment with each EGFR antisense oligonucleotide. The ability to inhibit GEO anchorage‐independent growth, however, varied among the EGFR antisense sequences with an IC50 ranging between 0.5 and 3.5 μM. Two of these antisense oligonucleotides targeting the regions between 2457–2476 and 614–4633 bases of the human EGFR mRNA have been modified as hybrid DNA/RNA mixed backbone oligonucleotides (MBO) to examine their anticancer properties in vivo. The 2 EGFR antisense MBOs retained the same biological properties of the fully phosphorothioate EGFR antisense oligonucleotides targeting the same EGFR mRNA sequences, such as blocking EGFR synthesis, inhibiting cell growth and enhancing programmed cell death in human cancer cell lines that express functional EGFRs. Furthermore, a potentiation in the growth inhibitory effect on GEO cancer cells was observed after treatment with these EGFR antisense MBOs in combination with cytotoxic drugs, including cisplatin, doxorubicin, paclitaxel, or topotecan. These results show the antiproliferative activity of specific EGFR antisense oligonucleotides and allow to identify novel EGFR antisense MBOs that deserve further evaluation as potential selective anticancer agents alone or in combination with cytotoxic drugs in human carcinomas that express functional EGFRs.

Cooperative Antitumor Effect of Multitargeted Kinase Inhibitor ZD6474 and Ionizing Radiation in Glioblastoma

Purpose: Glioblastoma multiforme is an aggressive disease in which vascular endothelial growth factor (VEGF) and the EGF receptor (EGFR) are implicated in tumor growth, relapse, and resistance to radiotherapy and chemotherapy. The VEGF receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR), typically present on endothelial cells, have also been identified in human glioblastoma tissues and cell lines. In addition, EGFR is dysregulated in the majority of human glioblastomas and EGFR overexpression correlates with shorter survival. We have investigated the antitumor and antiangiogenic effect of ZD6474, an inhibitor of both VEGFR and EGFR signaling as a single agent and in combination with ionizing radiation. Experimental Design: We have used ZD6474 and/or ionizing radiation in human glioblastoma cell lines D54 and U251 in vitro and in nude mice bearing established xenografts. The effects of treatment on tumor blood vessels and protein expression were evaluated by Western blot and immunohistochemistry. Results: As single agents, ionizing radiation and ZD6474 caused a dose-dependent inhibition of soft agar growth in D54 and U251 cell lines, whereas a cooperative effect was obtained in combination. Treatment of mice bearing D54 xenografts with either ZD6474 or radiotherapy alone caused tumor growth inhibition that was reversible upon treatment cessation. A cooperative and long-lasting inhibition of tumor growth was obtained with ZD6474 in combination with concomitant radiotherapy. The antiproliferative effect was accompanied by inhibition of VEGF protein expression and inhibition of angiogenesis as measured by vessel counting. Conclusion: This study shows the antitumor activity of ZD6474 in combination with ionizing radiation in glioblastoma both in vitro and in vivo, and provides a scientific rationale to evaluate ZD6474 alone or in combination with radiotherapy in patients affected by this disease.

Goserelin depot in the treatment of premenopausal advanced breast cancer

333 pre- and peri-menopausal patients with breast cancer entered a programme of open studies on the effect of goserelin. Of the 333 patients, 265 patients were entered into assessable efficacy studies. Efficacy data were analysed from 228 eligible patients receiving 3.6 mg of goserelin administered as a subcutaneous injection of a depot formulation once every 28 days. Mean serum luteinising hormone (LH) and oestradiol concentrations were suppressed by day 22 after the first injection. Subjective response occurred in 68.3% of patients assessed. Objective response (UICC criteria) occurred in 36.4% of patients and the lifetable median duration of response was 44 weeks. Responses were observed in all histological grades of tumour, and regardless of oestrogen receptor status. Treatment was well tolerated with no withdrawals due to possible adverse reactions of which hot flushes (75.9%) and loss of libido (47.4%) were commonly encountered. Goserelin provides an effective well tolerated medical alternative to ovarian ablation in the management of advanced breast cancer.

Novel Toll-Like Receptor 9 Agonist Induces Epidermal Growth Factor Receptor (EGFR) Inhibition and Synergistic Antitumor Activity with EGFR Inhibitors

Purpose: Immunostimulating Toll-like receptor 9 (TLR9) agonists cause antitumor activity interfering also with cancer proliferation and angiogenesis by mechanisms still incompletely understood. We hypothesized that modified TLR9 agonists could impair epidermal growth factor receptor (EGFR) signaling and, by this means, greatly enhance EGFR inhibitors effect, acting on both the receptor targeting and the immunologic arm. Experimental Design: We used a novel second-generation, modified, immunomodulatory TLR9 agonist (IMO), alone and in combination with the anti-EGFR monoclonal antibody cetuximab or tyrosine kinase inhibitor gefitinib, on the growth of GEO and cetuximab-resistant derivatives GEO-CR colon cancer xenografts. We have also evaluated the expression of several proteins critical for cell proliferation, apoptosis, and angiogenesis, including EGFR, mitogen-activated protein kinase, Akt, bcl-2, cyclooxygenase-2, vascular endothelial growth factor, and nuclear factor-κB. Results: IMO inhibited GEO growth and signaling by EGFR and the other proteins critical for cell proliferation and angiogenesis. IMO plus the anti-EGFR antibody cetuximab synergistically inhibited tumor growth, signaling proteins, and microvessel formation. EGFR signaling inhibition by IMO is relevant because IMO cooperated also with EGFR tyrosine kinase inhibitor gefitinib in GEO tumors, while it was inactive against GEO-CR xenografts. On the other hand, IMO boosted the non-EGFR-dependent cetuximab activity, causing a cooperative antitumor effect in GEO-CR cells. Finally, combination of IMO, cetuximab and chemotherapeutic irinotecan eradicated the tumors in 90% of mice. Conclusion: IMO interferes with EGFR-related signaling and angiogenesis and has a synergistic antitumor effect with EGFR inhibitors, especially with cetuximab, boosting both the EGFR dependent and independent activity of this agent. Moreover, this therapeutic strategy could be translated in patients affected by colorectal cancer.

A novel MDM2 anti-sense oligonucleotide has anti-tumor activity and potentiates cytotoxic drugs acting by different mechanisms in human colon cancer

MDM2 is over‐expressed in several human tumors. Its product is a negative‐feedback regulator of p53, which interferes with the control of cell proliferation and apoptosis, interacting not only with p53 but also with retinoblastoma (Rb) and E2F. Moreover, mutations in the ARF‐Ink4a locus may also allow MDM2 to override p53 functions. In this study, we have used a novel oligonucleotide anti‐sense MDM2, with mixed‐backbone structure and demonstrate that it causes inhibition of MDM2 expression, induction of both p53 and p21/WAF1 expression and a dose‐dependent, growth‐inhibitory effect in human GEO colon‐cancer cells. We also show that anti‐sense MDM2 has a co‐operative growth‐inhibitory effect with different classes of cytotoxic drugs acting by different mechanisms. Moreover, anti‐sense MDM2 induces apoptosis and markedly enhances the apoptotic activity of different cytotoxic drugs. Finally, we show that anti‐sense MDM2 has anti‐tumor activity in vivo in nude mice bearing GEO xenografts and potentiates the anti‐tumor effect of cytotoxic drugs. Indeed, despite the short treatment period, the combination of anti‐sense MDM2 and cytotoxic drugs causes a marked delay in tumor growth and prolongation of mice survival, lasting several months after treatment cessation. The anti‐tumor effect is associated with inhibition of MDM2 expression in tumor specimens of animals treated with anti‐sense MDM2, alone or in combination with a cytotoxic drug. Our results provide the rationale for development of a novel mixed‐backbone anti‐sense MDM2 into a clinical setting in therapeutic combination strategies with conventional cytotoxic drugs. Int. J. Cancer 88:804–809, 2000.