Elizabeth Zborowska

Demonstration That Mutation of the Type II Transforming Growth Factor β Receptor Inactivates Its Tumor Suppressor Activity in Replication Error-positive Colon Carcinoma Cells

Escape from negative growth regulation by transforming growth factor β (TGF-β) as a result of the loss of TGF-β type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-β treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-β-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-β autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Reduced Expression of Transforming Growth Factor β Type I Receptor Contributes to the Malignancy of Human Colon Carcinoma Cells

Transforming growth factor β (TGFβ) type I (RI) and type II (RII) receptors are essential for TGFβ signal transduction. A human colon carcinoma cell line, designated GEO, is marginally responsive to TGFβ and expresses a low level of RI mRNA relative to colon carcinoma cells, which are highly responsive to TGFβ. Hence, the role of RI as a limiting factor for TGFβ sensitivity and the contribution of low RI levels to the malignant phenotype of GEO cells were examined. Stable transfection of a tetracycline-regulatable rat RI cDNA increased TGFβ1 binding to RI and resulted in increased growth inhibition by exogenous TGFβ1. In contrast, although stable transfection of an RII expression vector into the same GEO cells increased TGFβ1 binding to RII, growth inhibition by exogenous TGFβ1 was not altered. This indicated that the low level of RI is a limiting factor for the growth-inhibitory effects of TGFβ in GEO cells. RI-transfected cells were growth-arrested at a lower saturation density than GEO control cells. They also showed reduced growth and clonogenicity in plating efficiency and soft agarose assays, whereas RII-transfected cells did not show any differences from the NEO control cells in these assays. Tetracycline repressed RI expression in transfected cells and reversed the reduction in plating efficiency of RI-transfected clones, confirming that growth effects were due to increased RI expression in transfected cells. TGFβ1 neutralizing antibody stimulated the proliferation of RI-transfected cells but had little effect on GEO control cells, indicating that increased autocrine-negative TGFβ activity also resulted from increased RI expression. Tumorigenicity in athymic nude mice was significantly delayed in RI-transfected cells. These results indicate that low RI expression can be a limiting factor for response to exogenous TGFβ, as well as TGFβ autocrine-negative activity, and that reduction of RI expression can contribute to malignant progression.

Blockade of EGFR and ErbB2 by the Novel Dual EGFR and ErbB2 Tyrosine Kinase Inhibitor GW572016 Sensitizes Human Colon Carcinoma GEO Cells to Apoptosis

Coexpression of the epidermal growth factor receptor (EGFR) family receptors is found in a subset of colon cancers, which may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. Recently, efforts have been made to develop novel 4-anilinoquinazoline and pyridopyrimidine derivatives to inhibit EGFR and ErbB2 kinases simultaneously. In this study, we tested the efficacy of a novel reversible dual inhibitor GW572016 compared with the selective EGFR and ErbB2 tyrosine kinase inhibitors (TKI) AG1478 and AG879 and their combination, using the human colon adenocarcinoma GEO mode. GEO cells depend on multiple ErbB receptors for aberrant growth. A synergistic effect on inhibition of cell proliferation associated with induction of apoptosis was observed from the combination of AG1478 and AG879. Compared with AG1478 or AG879, the single TKI compound GW572016 was a more potent inhibitor of GEO cell proliferation and was able to induce apoptosis at lower concentrations. Western blot analysis revealed that AG1478 and AG879 were unable to suppress both EGFR and ErbB2 activation as well as the downstream mitogen-activated protein kinase (MAPK) and AKT pathways as single agents. In contrast, GW572016 suppressed the activation of EGFR, ErbB2, MAPK, and AKT in a concentration-dependent manner. Finally, in vivo studies showed that GW572016 treatment efficiently blocked GEO xenograft growth at a dose range of 30 to 200 mg/kg with a twice-daily schedule. In summary, our study indicates that targeting both EGFR and ErbB2 simultaneously could enhance therapy over that of single agents directed at EGFR or ErbB2 in cancers that can be identified as being primarily heterodimer-dependent.

Synergistic efficacy of O6benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) repairs cytotoxic DNA damage formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). High levels of this repair protein cause tumor drug resistance to nitrosoureas. To investigate the ability of a direct alkyltransferase inhibitor, O6-benzylguanine, to reverse the nitrosourea resistance of human colon cancer cells, we studied the VACO 6 cell line which has high alkyltransferase and is completely resistant to BCNU at maximal tolerated doses in the xenograft model. O6-Benzylguanine at 0.5 microgram/mL for 1 hr inactivated VACO 6 alkyltransferase by > 98% and reduced the IC50 of BCNU by 3- to 4-fold. Further analysis indicated that these two agents act in a highly synergistic fashion. In xenograft bearing athymic mice, dose-dependent depletion of hepatic and tumor alkyltransferase was noted. To maintain alkyltransferase depletion in the xenograft for at least 24 hr, two doses of 60 mg/kg O6-benzylguanine were given 1 hr prior and 7 hr after BCNU. Under these conditions, VACO 6 xenografts became responsive to BCNU with significant reductions (P < 0.001) in the tumor growth rate. The combination increased toxicity to the host, reducing the maximum tolerated dose of BCNU by approximately 50%. This study provides definitive evidence that high alkyltransferase activity is responsible for BCNU resistance in human colon cancer xenografts and that with careful drug scheduling, O6-benzylguanine can sensitize a tumor which is completely unresponsive to BCNU alone. Further studies which optimize the therapeutic index of BCNU and O6-benzylguanine in vivo will define the schedule to be used in broader preclinical studies.

Control of Type II Transforming Growth Factor-β Receptor Expression by Integrin Ligation

Ectopic expression of the α5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of α5integrin is due to induction of autocrine negative transforming growth factor-β (TGF-β) activity. MCF-7 breast cancer cells do not express either α5 integrin or type II TGF-β receptor and hence are unable to generate TGF-β signal transduction. Ectopic expression of α5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-β receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in α5transfectants by growth on fibronectin-coated plates. Induction of type II TGF-β receptor expression also resulted in the generation of autocrine negative TGF-β activity because colony formation was increased after TGF-β neutralizing antibody treatment. Transient transfection with a TGF-β promoter response element in tandem with a luciferase cDNA into cells stably transfected with α5integrin resulted in basal promoter activities 5–10-fold higher than those of control cells. Moreover, when α5 transfectants were treated with a neutralizing antibody to either TGF-β or integrin α5, this increased basal promoter activity was blocked. Autocrine TGF-β activity also induced 3-fold higher endogenous fibronectin expression in α5 transfectants relative to that of control cells. Re-expression of type II receptor by α5 transfection also restored the ability of the cells to respond to exogenous TGF-β and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-β type II receptor expression can be controlled by α5β1 ligation and integrin signal transduction. Moreover, TGF-β and integrin signal transduction appear to cooperate in their tumor-suppressive functions.

Autocrine transforming growth factor provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor α (TGF-α) was ectopically expressed by stable transfection of a TGF-α cDNA under repressible tetracycline control. The TGF-α-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-α transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-α-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-α-transfected cells indicate that autocrine TGF-α functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-α was associated with increased malignant properties as TGF-α transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-α expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-α. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-α.

Expression of transforming growth factor-β receptor type II and tumorigenicity in human breast adenocarcinoma MCF-7 cells

To analyze transforming growth factor‐β (TGF‐β) response during MCF‐7 cell progression, early passage (MCF‐7E, <200 passage) and late passage (MCF‐7L, > 500 passage) cells were compared. MCF‐7E cells showed an IC50 of ∼10 ng/ml of TGF‐β1, whereas MCF‐7L cells were insensitive. MCF‐7E cells contained approximately threefold higher levels of TGF‐β receptor type II (TβRII) mRNA than MCF‐7L, but their TβRI levels were similar. MCF‐7E parental cells showed higher TβRII promoter activity than MCF‐7L cells, which could be attributed to changes in Sp1 nuclear protein levels. Receptor cross‐linking studies indicated that the cell surface receptor levels parallel mRNA levels in both cell lines. Limiting dilution clones of MCF‐7E cells were established to determine the heterogeneity of TβRII expression in this cell line, and they showed varying degrees of TβRII expression. Fibronectin was induced at higher levels in cells expressing higher TβRII levels. All three TGF‐β isoforms were detected in limiting dilution clones and parental cells, but TGF‐β1 was more abundant relative to TGF‐β2 or 3, and no correlation between TGF‐β isoform profile with TGF‐β sensitivity was found. MCF‐7L cells were tumorigenic and formed xenografts rapidly and progressively, whereas MCF‐7E parental and limiting dilution clonal cells showed transient tumor formation followed by regression. These results indicate that decreased TβRII transcription in breast cancer cells leads to a loss of TβRII expression, resulting in cellular resistance to TGF‐β which contributes to escape from negative growth regulation and tumor progression. J. Cell. Physiol. 176:424–434, 1998.