Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance. Significance: A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance.

Arginine vasopressin controls p27(Kip1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells.

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated ß-galactosidase (SA-ßGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187.

Autophagy buffers Ras-induced genotoxic stress enabling malignant transformation in keratinocytes primed by human papillomavirus.

Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRasG12V activity in E6E7-HPV-keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRasG12V.

Fibroblast Growth Factor 2 lethally sensitizes cancer cells to stress-targeted therapeutic inhibitors.

In malignant transformation, cellular stress-response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress-targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K-Ras-driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response-checkpoint inhibitors triggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK-ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress-targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies.

Vasopressin triggers senescence in K-ras transformed cells via RhoA-dependent downregulation of cyclin D1.

Arginine vasopressin (AVP), a vasoactive peptide hormone that binds to three G-protein coupled receptors (V1R, V2R, and V3R), has long been known to activate V1R and elicit mitogenesis in several cell types, including adrenal glomerulosa cells. However, in the mouse Y1 adrenocortical malignant cell line, AVP triggers not only a canonical mitogenic response but also novel RhoA-GTP-dependent mechanisms which downregulate cyclin D1, irreversibly inhibiting K-ras oncogene-driven proliferation. In Y1 cells, AVP blocks cyclin D1 expression, induces senescence-associated beta-galactosidase (SAbeta-Gal) and inhibits proliferation. However, ectopic expression of cyclin D1 renders Y1 cells resistant to both SAbeta-Gal induction and proliferation inhibition by AVP. In addition, ectopic expression of the dominant negative RhoAN19 mutant blocks RhoA activation, yielding Y1 cell sub-lines which are no longer susceptible to cyclin D1 downregulation, SAbeta-Gal induction, or proliferation inhibition by AVP. Furthermore, inhibiting RhoA with C3 exoenzyme protects Y1 cells from AVP proliferation inhibition and SAbeta-Gal induction. On the other hand, AVP treatment does not activate caspases 3 and 7, and the caspase inhibitor Ac-DEVD-CMK does not protect Y1 cells from proliferation inhibition by AVP, implying that AVP does not trigger apoptosis. These results underline a pivotal survival activity of cyclin D1 that protects K-ras oncogene-dependent malignant cells from senescence.

An Interdisciplinary Approach for Designing Kinetic Models of the Ras/MAPK Signaling Pathway.

We present in this article a methodology for designing kinetic models of molecular signaling networks, which was exemplarily applied for modeling one of the Ras/MAPK signaling pathways in the mouse Y1 adrenocortical cell line. The methodology is interdisciplinary, that is, it was developed in a way that both dry and wet lab teams worked together along the whole modeling process.

Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes.

Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions.

c-Myc protein is stabilized by fibroblast growth factor 2 and destabilized by ACTH to control cell cycle in mouse Y1 adrenocortical cells.

ACTH is the hormone known to control adrenal cortex function and maintenance in the intact animal but, in culture, it inhibits proliferation of adrenocortical cells from different mammalian species, a puzzle that has remained unsolved for nearly 30 years. In this paper we compare ACTH and fibroblast growth factor 2 (FGF2) antagonistic effects on the cell cycle in the Y1 cell line, a functional lineage of mouse adreno-cortical tumor cells. This cell line displays chronic high levels of c-Ki-Ras-GTP, high active constitutive levels of phosphatidylinositol 3-OH kinase/Protein Kinase B (PI3K/AKT) and low constitutive basal expression of c-Myc, which accounts for a minor deregulation of the cell cycle. In G0/G1-arrested Y1 cells, over-expression of the dominant negative mutant HaRasN17 drastically reduces c-Ki-Ras-GTP levels, eliminating basal c-Myc expression and basal S phase entry. PI3K/Akt seems to be the downstream pathway from c-Ki-ras for deregulation of c-Myc basal expression, since wortmannin abolishes c-Myc expression in serum-starved, G0/G1-arrested Y1 cells. FGF2 is a strong mitogen for Y1 cells, promoting -- in a manner dependent on the MEK/ERK pathway -- c-myc transcription induction, c-Myc protein stabilization and S phase entry in G0/G1-arrested Y1 cells. On the other hand, ACTH causes c-Myc protein destabilization, partially blocking S phase entry induced by FGF2, by a process dependent on the cAMP/protein kinase A (PKA) pathway. The whole pathway activated by ACTH to destabilize c-Myc protein in Y1 cells might comprise the following steps: ACTH receptor -->cAMP/PKA --> Akt deactivation -->GSK3 activity liberation --> c-Myc Thr58 phosphorylation. We demonstrate that c-Myc regulation is a central key in the cell cycle control by these factors, since enforced expression of c-Myc through the MycER chimera abrogates the ACTH inhibitory effect over FGF2-induced S phase entry.

Deconstructing the molecular mechanisms of cell cycle control in a mouse adrenocortical cell line: roles of ACTH.

This is a progress report of an attempt to deconstruct the signaling network underlying cell cycle control in the mouse Y1 adrenocortical cell line, aiming to uncover ACTH growth regulatory pathways. Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene. Despite this oncogenic lesion, Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the sequential events comprising the mitogenic response triggered by FGF2 in G0/G1-arrested Y1 cells: 1) activation of ERK1/2 and PI3K, by 5 minutes; 2) induction of c-Fos and c-Myc proteins by 2 hours; 3) induction of cyclin D1 protein by 5 hours; 4) phosphorylation of Rb protein between 6 and 8 hours; 5) onset of DNA synthesis by 8-9 hours. In this cell line, ACTH-receptor (ACTH-R) activates contradictory pathways of growth regulation. First, ACTH coordinately induces fos and jun gene families via activation of both ERK1/2 and cAMP/PKA pathways, resembling a mitogen. Second, ACTH-R triggers cAMP/PKA-mediated antimitogenic mechanisms comprised of Akt/PKB dephosphorylation/deactivation, c-Myc protein degradation, and p27(Kip1) protein induction. Induction of cyclin D1 depends on activation of both ERK1/2 and PI3K, but is not affected by ACTH action. As a consequence, ACTH antagonizes FGF2 mitogenic activity but ectopic expression of the c-Myc protein (via MycER fusion protein) is sufficient to abrogate this ACTH antagonistic effect over FGF2 mitogenic activity. Ectopic expression of both c-Myc and cyclin D1 is not sufficient to drive G0/G1-arrested Y1 cells into S phase, but when the sustained expression of these two proteins is complemented by ACTH treatment it promotes G1 phase progression and DNA synthesis initiation. In conclusion, ACTH-receptor lacks signaling potential sufficient to initiate a mitogenic response in Y1 adrenocortical cells and, therefore, cannot substitute for bona fide mitogens like FGF2.

Fibroblast growth factor 2 causes G2/M cell cycle arrest in ras-driven tumor cells through a Src-dependent pathway.

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3(Ras) mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

c-Ki-ras oncogene amplification and FGF2 signaling pathways in the mouse Y1 adrenocortical cell line.

The mouse Y1 adrenocortical tumor cell line is highly responsive to FGF2-(Fibroblast Growth Factor 2) and possesses amplified and over-expressed c-Ki-ras proto-oncogene. We previously reported that this genetic lesion leads to high constitutive levels of activation of the c-Ki-Ras-GTP-->PI3K-->Akt signaling pathway (Forti et al. 2002). On the other hand, activation levels of another important pathway downstream of c-Ki-Ras-GTP, namely, Raf-->MEK-->ERK, remain strictly dependent on FGF2 stimulation (Rocha et al. 2003). Here we show that, first, FGF2 transiently up-regulates the c-Ki-Ras-GTP-->PI3K-->Akt pathway, in spite of its high basal levels. Second, c-Ki-Ras-GTP transient up-regulation likely underlies activation of the ERK1/2 pathway by FGF2. Third, c-Ki-Ras-GTP high basal levels suppress activation of the c-H-Ras onco-protein. But, Y1 cells, expressing dominant negative mutant RasN17, display a rapid and transient up-regulation of c-H-Ras-GTP upon FGF2 treatment. Elucidation of FGF2-signaling pathways in Y1 tumor cells can uncover new targets for drug development of interest in cancer therapy.

Arginine vasopressin inhibition of cyclin D1 gene expression blocks the cell cycle and cell proliferation in the mouse Y1 adrenocortical tumor cell line.

Arginine vasopressin (AVP) is a nonapeptide long known as an endocrine and paracrine regulator of important systemic functions, namely, vasoconstriction, gluconeogenesis, corticosteroidogenesis, and excretion of water and urea. Here we report, for the first time, that AVP specifically inhibits expression of the cyclin D1 gene, leading to cell cycle blockage and halting cell proliferation. In G0/G1-arrested mouse Y1 adrenocortical tumor cells, maintained in serum-free medium (SFM), AVP mimics FGF2, promoting rapid ERK1/2 activation (5 min) followed by c-Fos protein induction (2 h). PKC inhibitor Go6983 and PI3K inhibitors wortmannin and LY294002 all inhibit ERK1/2 activation by AVP, but not by FGF2. Thus, AVP and FGF2 concur to activate ERK1/2 by different regulatory pathways. However, AVP is not a mitogenic factor for Y1 cells. On the contrary, AVP strongly antagonizes FGF2 late induction (2-5 h) of the cyclin D1 gene, down-regulating both cyclin D1 mRNA and protein. AVP inhibition of cyclin D1 expression is sufficient to block G1 phase progression and cell entry into the S phase, monitored by BrdU nuclear labeling. In addition, AVP completely inhibits proliferation of Y1 cells in 10% fetal calf serum (10% FCS) medium. On the other hand, ectopic expression of the cyclin D1 protein renders Y1 cells resistant to AVP for both entry into the S phase in SFM and continuous proliferation in 10% FCS medium. In conclusion, inhibition of cyclin D1 expression by AVP is an efficient mechanism of cell cycle blockage and consequent proliferation inhibition in Y1 adrenocortical cells.

Molecular mechanisms of cell cycle control in the mouse Y1 adrenal cell line.

Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene, displaying chronic high levels of the c-Ki-Ras-GTP protein. Despite this oncogenic lesion, we previously reported that Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the mitogenic response triggered by FGF2 in G0/G1-arrested cells. ACTH, on the other hand, elicits cAMP/PKA-mediated antimitogenic mechanisms involving Akt/PKB dephosphorylation/deactivation and c-Myc protein degradation, blocking G1 phase progression stimulated by FGF2. In this paper we report that ACTH does not directly antagonize any of the early or late sequential steps comprising the mitogenic response triggered by FGF2. In effect, ACTH targets deactivation of constitutively phosphorylated-Akt, restraining the potential of c-Ki-Ras-GTP to subvert Y1 cell cycle control. Thus, we can consider ACTH a tumor suppressor rather than an antimitogenic hormone. In addition, we present initial results showing that high constitutive levels of c-Ki-Ras-GTP render Y1 cells susceptible to dye upon FGF2 treatment. This surprising FGF2 death-effect, that is independent of the well known FGF2-mitogenic activity, might involve a natural unsuspected mechanism for restraining oncogene-induced proliferation.

ACTH promotion of p27(Kip1) induction in mouse Y1 adrenocortical tumor cells is dependent on both PKA activation and Akt/PKB inactivation.

Here we report antimitogenic mechanisms activated by the adrenocorticotropic hormone (ACTH) in the mouse Y1 adrenocortical tumor cell line. ACTH receptors activate the Galphas/adenylate cyclase cAMP/PKA pathway to promote dephosphorylation of Akt/PKB enzymes, leading to induction of the cyclin-dependent kinases' (CDKs) inhibitor p27(Kip1). Y1 cells display high constitutive levels of phosphorylated Akt/PKB dependent on chronically elevated c-Ki-Ras.GTP and PI3K activity. Expression of the dominant negative mutant RasN17 in Y1 cells results in strong reduction of both c-Ki-Ras.GTP and phosphorylated Akt/PKB, which are restored by FGF2 treatments. Inhibitors of PI3K lead to rapid dephosphorylation of Akt/PKB and block phosphorylation of Akt/PKB promoted by FGF2. ACTH rapidly promotes dephosphorylation of Akt/PKB in Y1 adrenal cells, while constitutively high levels of c-Ki-Ras.GTP remain unchanged. ACTH and cAMP elevating agents fail to cause Akt/PKB dephosphorylation in PKA-deficient clonal mutants of Y1 cells. In addition, cholera toxin, forskolin, and 8BrcAMP all mimic ACTH, causing dephosphorylation of Akt/PKB in wild-type Y1 cells. ACTH is unable to prevent Akt/PKB phosphorylation, promoted by FGF2 in clonal lines of RasN17-Y1 transfectants displaying negligible levels of c-Ki-Ras.GTP. ACTH promotes strong p27(Kip1) protein induction in wild-type Y1 adrenocortical cells but not in PKA-deficient Y1-clonal mutants nor in RasN17-Y1 transfectants. PI3K inhibitors induce p27(Kip1) protein in all cells studied, i.e., wild type and transfectants. The inverse correlation between levels of phosphorylated Akt/PKB and of p27(Kip1) protein caused by ACTH suggests a novel antimitogenic pathway activated by ACTH and mediated by cAMP/PKA in the mouse Y1 adrenocortical tumor cell line.

c-Ki-ras oncogene amplification and FGF2 signaling pathways in the mouse Y1 adrenocortical cell line

A linhagem tumoral Y1, originada de adrenocórtex decamundongo responde a FGF2 (Fator de Crescimento de Fibroblasto), possui o proto-oncogene c-ki-ras amplificado e a proteína c-Ki-Ras super-expressa e ativa (c-Ki-Ras-GTP). Em trabalhos anteriores mostramos que esta lesão genética causa ativação constitutiva da via de sinalização: c-Ki-Ras-GTP®PI3K®Akt (Forti et al. 2002). Por outro lado, a ativação da via de Raf® MEK®ERK, permanece estritamente dependente de estímulos de FGF2 (Rocha et al. 2003). Neste trabalho mostramos, primeiro, que estímulos de FGF2 ativam transientemente a via c-Ki-Ras-GTP®PI3K®Akt para níveis superiores aos expressos constitutivamente. Segundo, a ativação transiente de c-Ki-Ras-GTP por FGF2 permite a ativação da via de ERK1/2. Terceiro, os níveis basais elevados de c-Ki-Ras-GTP inibem a ativação da proteína c-H-Ras, pois células Y1 expressando o mutante negativo RasN17 apresentam uma rápida e transiente ativação de c-H-Ras-GTP após tratamentos de FGF2. Estes estudos das vias de sinalização acionadas por FGF2 em células adrenais tumorais Y1 podem fornecer novos alvos para o desenvolvimento de drogas de interesse para terapia oncogênica.

Regulation of G0 G1 S transition: a genetic approach

A genetic approach was adopted to analyze the cell cycle G(O)(G (1) (S transition in mouse Balb/ 3T3 fibroblasts (clone A3l). We designed selection procedures to isolate revertant from the EJ-ras transformed Balb/3T3 ribroblasts that had recovered strict -control of the G(O) ( G(1), transition by serum growth factors. The aim was to uncover phenotypic traits associated with malignancy (high growth rate G(1) phase shortening and high tumorigenicity) that segregate independently.

Induction of fos and jun proto-oncogenes by the adrenocorticotropic hormone (ACTH): possible biological implications

ACTH induces the expression of fos and jun proto-oncogene family members in the mouse Y-1 adrenocortical cell fine. PMA (phorbol-12-myristate-l3-acetate) closely mimics these inductive effects of ACTH. On the other hand, cAMP derivatives are not effective in inducing the fos and jun genes. These results suggest that ACTH receptors are likely to activate signaling routes other than the classical cAMP/protein kinase A in order to induce FOS and JUN proteins. We hypothesize that induction of FOS and JUN proteins is likely to be important in the trophic response of adrenocortical cells to ACTH.