Search

The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of <10% due in part to a lack of effective therapies. Pan-histone deacetylase (HDAC) inhibitors have shown preclinical efficacy against PDAC but have failed in the clinic due to toxicity. Selective HDAC inhibitors may reduce toxicity while retaining therapeutic efficacy. However, their use requires identification of the specific HDACs that mediate the therapeutic effects of HDAC inhibitors in PDAC. We determined that the HDAC1/2/3 inhibitor Mocetinostat synergizes with the HDAC4/5/6 inhibitor LMK-235 in a panel of PDAC cell lines. Furthermore, while neither drug alone synergizes with gemcitabine, the combination of Mocetinostat, LMK-235, and gemcitabine showed strong synergy. Using small interfering (si)RNA-mediated knockdown, this synergy was attributed to inhibition of HDACs 1, 2, and 6. Pharmacological inhibition of HDACs 1 and 2 with Romidepsin and HDAC6 with ACY-1215 also potently synergized with gemcitabine in a panel of PDAC cell lines, and this drug combination potentiated the antitumor effects of gemcitabine against PDAC xenografts in vivo. Collectively, our data show that inhibition of multiple HDACs is required for therapeutic effects of HDAC inhibitors and support the development of novel strategies to inhibit HDACs 1, 2, and 6 for PDAC therapy.

Recepteur d'origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer.

This study identifies a novel cross-talk paradigm between the type I insulin-like growth factor receptor (IGF1R) and epidermal growth factor receptor (EGFR) in colon cancer cells. IGF1R activation by ligand exposure in growth factor-deprived cells induces Akt activation in the FET, CBS, and GEO colon cancer cell lines. Investigation of IGF1R-mediated signaling pathways using small interfering RNA approaches indicated that, as expected, phosphatidylinositol 3'-kinase (PI3K) was activated by IGF1R. Mitogen-activated protein kinase (MAPK) activity as reflected by phospho-extracellular signal-regulated kinase (ERK) induction was not significantly activated until later times following release of these cells from growth factor deprivation stress. The appearance of phospho-ERK was proximal to EGFR activation. Treatment of cells with the PI3K inhibitor LY294002 before release from stress resulted in a concentration-dependent loss of EGFR activation, whereas treatment with the MAPK inhibitor PD98059 did not block EGFR activation, indicating that EGFR activation was downstream of the IGF1R/PI3K pathway. PD98059 inhibition of MAPK was associated with a concentration-dependent reduction in EGFR-mediated phospho-ERK. EGFR inhibitor blocked induction of phospho-ERK, showing that MAPK activity was a consequence of EGFR-mediated signaling. On the other hand, a small-molecule IGF1R inhibitor, PQIP, blocked Akt phosphorylation. The divergent signaling functions of IGF1R and EGFR suggested the potential for synergism by a combination of therapy directed at the two receptors. Combination treatment with PQIP and EGFR inhibitor Tarceva resulted in synergistic effects as indicated by combination index analysis in all three cell lines tested.

The role of the ErbB family in supporting the malignant phenotype was characterized by stable transfection of a single chain antibody (ScFv5R) against ErbB2 containing a KDEL endoplasmic reticulum retention sequence into GEO human colon carcinoma cells. The antibody traps ErbB2 in the endoplasmic reticulum, thereby down-regulating cell surface ErbB2. The transfected cells showed inactivation of ErbB2 tyrosine phosphorylation and reduced heterodimerization of ErbB2 and ErbB3. This resulted in greater sensitivity to apoptosis induced by growth deprivation and delayed tumorigenicity in vivo. Furthermore, decreased heterodimerization of ErbB2 and ErbB3 led to a reorganization in ErbB function in transfected cells as heterodimerization between epidermal growth factor receptor (EGFR) and ErbB3 increased, whereas ErbB3 activation remained almost the same. Importantly, elimination of ErbB2 signaling resulted in an increase in EGFR expression and activation in transfected cells. Increased EGFR activation contributed to the sustained cell survival in transfected cells.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.

Recently, we have shown that autocrine transforming growth factor-alpha (TGF-alpha) controls the expression of integrin alpha2, cell adhesion to collagen IV and motility in highly progressed HCT116 colon cancer cells (Sawhney, R. S., Zhou, G-H. K., Humphrey, L. E., Ghosh, P., Kreisberg, J. I., and Brattain, M. G. (2002) J. Biol. Chem. 277, 75-86). We now report that expression of basal integrin alpha2 and its biological effects are controlled by constitutive activation of the extracellular signal-regulated/mitogen-activated protein kinase (ERK/MAPK) pathway. Treatment of cells with selective mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 and U0126 showed that integrin alpha2 expression, cell adhesion, and activation of ERK are inhibited in a parallel concentration-dependent fashion. Moreover, autocrine TGF-alpha-mediated epidermal growth factor receptor activation was shown to control the constitutive activation of the ERK/MAPK pathway, since neutralizing antibody to the epidermal growth factor receptor was able to block basal ERK activity. TGF-alpha antisense-transfected cells also showed attenuated activation of ERK. Using a real time electric cell impedance sensing technique, it was shown that ERK-dependent integrin alpha2-mediated cell micromotion signaling is controlled by autocrine TGF-alpha. Thus, this study implicates ERK/MAPK signaling activated by endogenous TGF-alpha as one of the mechanistic features controlling metastatic spread.

The inappropriate expression of TGFalpha in growth arrest contributes to malignant progression in human colon carcinoma cells. Early stage, non-progressed colon tumor cells show a down-regulation of TGFalpha in growth arrest and require both nutrients and growth factors for re-entry into the cell cycle. In contrast, highly progressed cells up-regulate TGFalpha during growth arrest and require only nutrients for re-entry. Given the importance of TGFalpha in malignant progression, this work addressed the regulation of TGFalpha expression in the early stage colon carcinoma cell line, FET. Growth-arrested FET cells down-regulated the expression of TGFalpha, EGFr and, in turn, EGFr activation. These quiescent cells continued to express high levels of IGF-IR protein, but IGF-IR activation was undetectable. Cell cycle re-entry required exogenous growth factor activation of the IGF-IR by insulin or IGF-I. This IGF-IR activation resulted in S phase re-entry and was accompanied by an approximate threefold induction of TGFalpha expression along with EGFr activation at 1 h following release from growth arrest. Activation of IGF-IR occurred within 5 min of cell-cycle re-entry. Previously identified DNA binding proteins which bind to a unique TGFalpha/EGF response element within the TGFalpha promoter were similarly induced following IGF-IR activation. The addition of EGFr neutralizing antibodies abolished the activated IGF-IR stimulated S phase re-entry. Moreover, disruption of the growth arrest associated down-regulation of TGFalpha in FET cells by constitutive TGFalpha expression abrogated the requirement for IGF-IR activation for cell cycle re-entry. Consequently, this study indicates, for the first time, that IGF-IR activation up-regulates components of the TGFalpha autocrine loop resulting in TGFalpha-mediated EGFr activation which was critical for IGF-IR mediated re-entry into the cell cycle from the growth-arrested state.

Despite constitutive expression of autocrine transforming growth factor-alpha (TGF-alpha) in growth factor-independent colon carcinoma cells, the epidermal growth factor receptor (EGFr) is not saturated and can be further activated by exogenous EGFr ligand. Given that the activation of EGFr by exogenous growth factor has no further effect on DNA synthesis, the question arises as to what function this additional EGFr activation might have. We report that EGF induces integrin alpha2 expression, integrin-mediated adhesion, and micromotility of HCT116 cells. The stimulatory effect of ligand on these biological functions is abrogated by treatment with AG1478- and EGFr-blocking monoclonal antibody. This provides evidence that the biological responses are EGFr-mediated and EGFr is located upstream of integrin alpha2 expression. Therefore, although exogenous EGF has no effect on DNA synthesis beyond that induced by autocrine TGF-alpha (at subsaturating levels of EGFr occupation) exogenous growth factor does induce integrin alpha2 expression, cell adhesion, and micromotion. An important finding revealed by this study is the documentation of biological responses of EGFr-mediated functions, including DNA synthesis, cell adhesion, and micromotion, which differ in sensitivity with respect to different degrees of EGFr activation at the basal state and in response to exogenous ligand.

Autocrine TGFalpha is an important growth effector in the transformed phenotype. Growth stimulation of some colon cancer cells as well as other types of cancer cells is effected by activation of the epidermal growth factor receptor. Importantly, this receptor activation leads to further stimulation of TGFalpha transcription and increased peptide synthesis. However, the molecular mechanism by which TGFalpha transcription is activated is poorly understood. In this paper, we describe the localization of a cis-sequence within the TGFalpha promoter which mediates this stimulation. This region contains parallel cis-acting elements which interact to regulate both basal and EGF-induced TGFalpha expression. The well differentiated colon carcinoma cell line designated FET was employed in these studies. It produces autocrine TGFalpha but requires exogenous EGF in the medium for optimal growth. Addition of EGF to FET cells maintained in the absence of EGF resulted in a 2 - 3-fold increase of both TGF promoter activity and endogenous TGFalpha mRNA at 4 h. This addition of EGF also stimulated protein synthesis. The use of deletion constructs of the TGFalpha promoter in chimeras with chloramphenicol acetyl transferase localized EGF-responsiveness to between -247 and -201 within the TGFalpha promoter. A 25 bp sequence within this region conferred EGF-responsiveness to heterologous promoter constructs. Further use of deletion/mutation chimeric constructs revealed the presence of at least two interacting cis-elements, one binding a repressor activity and the other, an activator. Gel shift studies indicate the presence of distinct complexes representing activator and repressor binding, which are positively modulated by EGF. The type and amount of complexes formed by these proteins interact to regulate both the basal activity and EGF-responsiveness of the TGFalpha promoter. The interaction of an activator protein with an EGF-responsive repressor may serve to regulate the level of this progression-associated, transforming protein within tight limits.

Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.

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 alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-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-alpha 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-alpha-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-alpha-transfected cells indicate that autocrine TGF-alpha 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-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha 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-alpha. 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-alpha.

Autocrine transforming growth factor alpha (TGFalpha) activity and control mechanisms for its expression were examined in a representative clonal isolate (CBS4) of a well-differentiated human colon carcinoma cell line designated CBS. CBS4 cells expressed TGFalpha and its receptor, epidermal growth factor receptor (EGFr). Blockade of EGFr and TGFalpha by neutralizing antibodies inhibited clonal growth and the initiation of DNA synthesis from quiescence in CBS4 cells. Therefore, TGFalpha is an autocrine growth factor for CBS4 cells. Several studies have indicated that activation of the EGFr by exogenous EGF stimulates TGFalpha expression. However, in CBS4 cells EGF did not induce TGFalpha mRNA expression, indicating that EGF does not affect TGFalpha transcription in these cells. Exogenous treatment of exponentially growing cells with either EGF or EGFr blocking antibody enhanced release of TGFalpha protein into the conditioned medium. This indicated that the release of TGFalpha into the conditioned medium by exogenous EGF was at least partially due to the displacement of TGFalpha from the TGFalpha/EGFr complexes. Similarly to exponentially growing cells, the EGFr blocking antibody and EGF also enhanced TGFalpha release into the medium of CBS4 cells after release from quiescence. These results indicated that exogenous EGF had little if any effect on TGFalpha expression in these cells and suggested that TGFalpha expression might be under endogenous TGFalpha control. Blockade of the autocrine TGFalpha loop by TGFalpha neutralizing antibody suppressed TGFalpha mRNA both in exponentially growing and quiescent cells, demonstrating that autocrine TGFalpha is autoregulatory in this system.

Autocrine transforming growth factor alpha (TGFalpha) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFalpha confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFalpha mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFalpha mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at -201 to -225 within the TGFalpha promoter which mediates the differential regulation of TGFalpha expression during quiescence establishment in these two cell lines. This same sequence confers TGFalpha promoter responsiveness to exogenous growth factor or autocrine TGFalpha. The abberant upregulation of TGFalpha mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFalpha approaches showed that the dysregulated TGFalpha expression in quiescent HCT116 cells is a function of the strong TGFalpha autocrine loop (not inhibited by blocking antibodies) in these cells.

Aberrant transcriptional regulation of transforming growth factor alpha (TGF alpha) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGF alpha expression in the malignant phenotype. In this paper, we report on TGF alpha promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGF alpha and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGF alpha. However, constitutive expression of TGF alpha antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGF alpha mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGF alpha autocrine activation is the major stimulator of TGF alpha expression in this cell line, TGF alpha promoter activity should be reduced in the antisense TGF alpha clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGF alpha promoter was restored in an antisense-TGF alpha-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system, we were able to identify a 25-bp element within the TGF alpha promoter which conferred TGF alpha autoregulation to the TGF alpha promoter in the HCT116 cell line. In the TGF alpha-antisense-RNA-expressing clones, this element was activated by exogenous EGF. This 25-bp sequence contained no consensus sequences of known transcription factors so that the TGF alpha or EGF regulatory element within this 25-bp sequence represents a unique element. Further characterization of this 25-bp DNA sequence by deletion analysis revealed that regulation of TGF alpha promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall expression of the activators is pivotal in determining the level of response to EGF or TGF alpha stimulation. The specific nuclear proteins binding to this region are also regulated in an autocrine-TGF alpha-dependent fashion and by exogenous EGF in EGF-deprived TGF alpha antisense clone 33. This regulation is identical to that seen in the growth factor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation of trans-acting factor binding by EGF suggests that the effect is directly due to growth factor and not mediated by changes in growth state. We conclude that this element appears to represent the major positive regulator of TGF alpha expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of TGF alpha promoter activity in the growth factor-independent phenotype.

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.

Although a great deal is known about the cellular effects of exogenous transforming growth factor-beta (TGF-beta) treatment and the effects of various exogenous agents (including TGF-beta's themselves) on TGF-beta expression, studies of cellular controls for autocrine TGF-beta expression and function have been rare. Since exogenous TGF-beta treatment blocks progression through the cell cycle, it seemed likely that autocrine TGF-beta activity would be induced by growth states in which there was little or no cell division such as confluency or quiescence. Specific TGF-beta 1 or beta 2 neutralizing antibody treatment of a colon carcinoma cell line designated CBS showed that autocrine TGF-beta activity could be demonstrated in quiescent cells but not in preconfluent cells. Studies of kinetics of TGF-beta 1 and beta 2 mRNA levels during the establishment of quiescence revealed a significant increase of both isoforms in quiescent cells. The quiescent cells also secreted three- to fourfold and four-to fivefold higher levels of total (latent plus active) TGF-beta 1 and beta 2 protein in the conditioned media than the confluent cells and preconfluent cells, respectively. There was no detectable active form of either TGF-beta isoform in the conditioned media of preconfluent cells, whereas a significant amount of active TGF-beta 1 and beta 2 was detected in the conditioned media of quiescent cells. Quantitative RNase protection assays were developed to compare the effects of cell crowding vs quiescence on TGF-beta expression. TGF-beta 1 was primarily induced by quiescence. TGF-beta 2 was induced by both quiescence and cell crowding. Increased TGF-beta 1 mRNA levels appeared to be exclusively due to an increase in stability, while increased TGF-beta 2 mRNA levels were due to increased transcription. This growth state-related induction of TGF-beta's was also observed in two other colon carcinoma cell lines. These studies show that TGF-beta 1 and beta 2 are autocrine-negative factors which can be situationally expressed by cells as a function of their growth state. Autocrine expression of the TGF-beta's in this model system appears not to affect exponentially growing cells, but rather to function by maintaining a quiescent state and/or by blocking progression through the cell cycle.

Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression.

Previously, we described a model culture system for comparing responsiveness of poorly differentiated and well-differentiated human colon carcinoma cells to exogenous growth factors. While polypeptide growth stimulators elicited an up-regulation of c-myc, as well as a mitogenic response in the well-differentiated cells, the poorly differentiated cells were insensitive to exogenous growth stimulators. We now show, by thymidine incorporation experiments and autoradiographic analysis, that transforming growth factor beta 1 (TGF-beta) abrogated the mitogenic responses to the growth factors epidermal growth factor + insulin + transferrin (IC50 = 0.8 ng/ml), as well as to nutrients (basal medium; IC50 = 0.2 ng/ml) in the well-differentiated cells. The poorly differentiated cells did not respond to TGF-beta. Moreover, TGF-beta (10 ng/ml) completely abrogated the growth factor-stimulated up-regulation of c-myc in the TGF-beta responsive, well-differentiated colon carcinoma cells. Addition of TGF-beta to the TGF-beta-responsive, well-differentiated cells, at a time after c-myc had been transiently up-regulated in response to growth stimulatory factors, resulted in a loss of responsiveness to TGF-beta. Addition of TGF-beta to these cells at increasing time periods after EIT stimulation also resulted in a loss of the TGF-beta-induced repression of c-myc. The results suggest an important role for c-myc in the mechanism of action of TGF-beta in well-differentiated human colon carcinoma cells.

Previously, we reported that exponentially proliferating cultures of well-differentiated human colon carcinoma cells responded to transforming growth factor beta 1 (TGF-beta) with growth inhibition, alterations in morphology, and increased secretion of the differentiation marker, carcinoembryonic antigen. Poorly differentiated cultures were unresponsive. Here we show that TGF-beta was ineffective in repressing nutrient-stimulated mitogenesis in quiescent, poorly differentiated cells. However, in quiescent, well-differentiated cells, TGF-beta repressed the mitogenic responses to both nutrients alone (by 90%) and to nutrients plus the exogenous stimulatory factors epidermal growth factor (E), insulin (I), and transferrin (T) (by 55-65%). Thymidine incorporation experiments indicated that TGF-beta reduced both the onset and peak mitogenic response to growth factors and/or nutrients in the well-differentiated cells. Additionally, TGF-beta repressed the growth factor (E + I + T)-stimulated upregulation of expression of both c-myc and of transforming growth factor alpha (TGF-alpha) mRNAs in quiescent, well-differentiated cells. TGF-beta also elicited a rapid (t1/2 approximately 1h) down-regulation of c-myc expression in the absence of prior growth factor (E + I + T) stimulation. In contrast, TGF-beta had no effect on c-myc or TGF-alpha mRNA expression in the poorly differentiated cells. The results suggest that TGF-beta exerts rapid inhibitory effects on proliferation-associated genes in quiescent and restimulated, well-differentiated cells. Expression of these genes (c-myc and TGF-alpha) may otherwise (in the absence of TGF-beta) play roles in the cellular signaling of mitogenic responses by growth stimulatory factors in well-differentiated colon carcinoma cells.