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Combined Inactivation of MYC and K-Ras oncogenes reverses tumorigenesis in lung adenocarcinomas and lymphomas.

Tran PT, Fan AC, Bendapudi PK, Koh S, Komatsubara K, Chen J, Horng G, Bellovin DI, Giuriato S, Wang CS, Whitsett JA, Felsher DW - PLoS ONE (2008)

Bottom Line: Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors.We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT

Background: Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as "oncogene-addiction." However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.

Methodology/principal findings: To examine how the MYC and K-ras(G12D) oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras(G12D) to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras(G12D)- or MYC/K-ras(G12D)-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras(G12D)-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras(G12D) resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras(G12D) in maintenance of lung tumors, we found that the down-stream mediators of K-ras(G12D) signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras(G12D) but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras(G12D). Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.

Conclusions/significance: Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.

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Conditional expression of MYC/K-rasG12D in the lung predisposes to bronchiogenic adenocarcinomas.(A) Double MYC/K-rasG12D (CMR)-induced tumors have histology consistent with adenomas/adenocarcinomas similar to MYC- and K-rasG12D-induced tumors on H&E. (B) To rule out the possibility that the double oncogene-induced lung tumors had developed doxycycline (or TetO)-dysregulated MYC or K-rasG12D expression, inactivated double oncogene-induced lung tumors were examined for spurious expression of MYC and K-rasG12D at the mRNA and/or protein level. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated (doxycycline removed from drinking water) for 2–9 weeks demonstrated lack of expression of the MYC transgene in contrast to a MYC-induced tumor that had never been inactivated. Immunohistochemical analysis (performed like Figure 1D) on similar (C) inactivated double oncogene-induced tumors also showed lack of MYC transgene product and endogenous murine MYC protein compared to a (D) MYC-activated tumor. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated demonstrated no expression of the (E) K-rasG12D transgene or upregulation of (F) endogenous murine K-ras (> = 3–9 tumors for > = 3 mice per experiment).
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pone-0002125-g004: Conditional expression of MYC/K-rasG12D in the lung predisposes to bronchiogenic adenocarcinomas.(A) Double MYC/K-rasG12D (CMR)-induced tumors have histology consistent with adenomas/adenocarcinomas similar to MYC- and K-rasG12D-induced tumors on H&E. (B) To rule out the possibility that the double oncogene-induced lung tumors had developed doxycycline (or TetO)-dysregulated MYC or K-rasG12D expression, inactivated double oncogene-induced lung tumors were examined for spurious expression of MYC and K-rasG12D at the mRNA and/or protein level. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated (doxycycline removed from drinking water) for 2–9 weeks demonstrated lack of expression of the MYC transgene in contrast to a MYC-induced tumor that had never been inactivated. Immunohistochemical analysis (performed like Figure 1D) on similar (C) inactivated double oncogene-induced tumors also showed lack of MYC transgene product and endogenous murine MYC protein compared to a (D) MYC-activated tumor. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated demonstrated no expression of the (E) K-rasG12D transgene or upregulation of (F) endogenous murine K-ras (> = 3–9 tumors for > = 3 mice per experiment).

Mentions: We were surprised to find that CM tumors were independent of MYC, since a multitude of previous studies have demonstrated that MYC-induced tumors exhibit complete tumor regression upon MYC inactivation [13]–[15], [17], [21], [22]. Studies demonstrated a subset of breast tumors induced by MYC overexpression that fail to undergo sustained regression upon MYC inactivation have mutations in K-Ras [16], [17]. To evaluate in our conditional lung model if MYC and K-Ras cooperate, we utilized a conditional mutant K-rasG12D line previously described, CCSP-rtTA/TetO-K-rasG12D (now called CR) [23]. CM and CR were then used to produce the bi-conditional animals CCSP-rtTA/TetO- MYC/TetO-K-rasG12D (or CMR), which upon doxycycline administration simultaneously overexpress both oncogenes under the control of the CCSP promoter in lung and as described below in lung tumors (Figure S2). At 3–4 weeks of age cohorts of CM, CR and CMR mice were treated with doxycycline and screened using physical exam and µCT screening. As described above, CM mice developed lung tumors with a median latency of 52 weeks (Figure 3A). Upon doxycycline treatment, CR mice developed lung adenocarcinomas with a median latency of 26 weeks. Surprisingly, CMR mice developed lung adenomas and adenocarcinomas (Figure 4A) with a latency of 36 weeks similar to the CR mice (Figure 3A; not significantly different by log-rank analysis, p>0.05). Thus, in the setting of adult lung epithelium MYC and K-rasG12D failed to cooperate to induce accelerated tumorigenesis.


Combined Inactivation of MYC and K-Ras oncogenes reverses tumorigenesis in lung adenocarcinomas and lymphomas.

Tran PT, Fan AC, Bendapudi PK, Koh S, Komatsubara K, Chen J, Horng G, Bellovin DI, Giuriato S, Wang CS, Whitsett JA, Felsher DW - PLoS ONE (2008)

Conditional expression of MYC/K-rasG12D in the lung predisposes to bronchiogenic adenocarcinomas.(A) Double MYC/K-rasG12D (CMR)-induced tumors have histology consistent with adenomas/adenocarcinomas similar to MYC- and K-rasG12D-induced tumors on H&E. (B) To rule out the possibility that the double oncogene-induced lung tumors had developed doxycycline (or TetO)-dysregulated MYC or K-rasG12D expression, inactivated double oncogene-induced lung tumors were examined for spurious expression of MYC and K-rasG12D at the mRNA and/or protein level. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated (doxycycline removed from drinking water) for 2–9 weeks demonstrated lack of expression of the MYC transgene in contrast to a MYC-induced tumor that had never been inactivated. Immunohistochemical analysis (performed like Figure 1D) on similar (C) inactivated double oncogene-induced tumors also showed lack of MYC transgene product and endogenous murine MYC protein compared to a (D) MYC-activated tumor. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated demonstrated no expression of the (E) K-rasG12D transgene or upregulation of (F) endogenous murine K-ras (> = 3–9 tumors for > = 3 mice per experiment).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365560&req=5

pone-0002125-g004: Conditional expression of MYC/K-rasG12D in the lung predisposes to bronchiogenic adenocarcinomas.(A) Double MYC/K-rasG12D (CMR)-induced tumors have histology consistent with adenomas/adenocarcinomas similar to MYC- and K-rasG12D-induced tumors on H&E. (B) To rule out the possibility that the double oncogene-induced lung tumors had developed doxycycline (or TetO)-dysregulated MYC or K-rasG12D expression, inactivated double oncogene-induced lung tumors were examined for spurious expression of MYC and K-rasG12D at the mRNA and/or protein level. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated (doxycycline removed from drinking water) for 2–9 weeks demonstrated lack of expression of the MYC transgene in contrast to a MYC-induced tumor that had never been inactivated. Immunohistochemical analysis (performed like Figure 1D) on similar (C) inactivated double oncogene-induced tumors also showed lack of MYC transgene product and endogenous murine MYC protein compared to a (D) MYC-activated tumor. qRT-PCR analysis of double oncogene-induced lung tumors from CMR mice that had been inactivated demonstrated no expression of the (E) K-rasG12D transgene or upregulation of (F) endogenous murine K-ras (> = 3–9 tumors for > = 3 mice per experiment).
Mentions: We were surprised to find that CM tumors were independent of MYC, since a multitude of previous studies have demonstrated that MYC-induced tumors exhibit complete tumor regression upon MYC inactivation [13]–[15], [17], [21], [22]. Studies demonstrated a subset of breast tumors induced by MYC overexpression that fail to undergo sustained regression upon MYC inactivation have mutations in K-Ras [16], [17]. To evaluate in our conditional lung model if MYC and K-Ras cooperate, we utilized a conditional mutant K-rasG12D line previously described, CCSP-rtTA/TetO-K-rasG12D (now called CR) [23]. CM and CR were then used to produce the bi-conditional animals CCSP-rtTA/TetO- MYC/TetO-K-rasG12D (or CMR), which upon doxycycline administration simultaneously overexpress both oncogenes under the control of the CCSP promoter in lung and as described below in lung tumors (Figure S2). At 3–4 weeks of age cohorts of CM, CR and CMR mice were treated with doxycycline and screened using physical exam and µCT screening. As described above, CM mice developed lung tumors with a median latency of 52 weeks (Figure 3A). Upon doxycycline treatment, CR mice developed lung adenocarcinomas with a median latency of 26 weeks. Surprisingly, CMR mice developed lung adenomas and adenocarcinomas (Figure 4A) with a latency of 36 weeks similar to the CR mice (Figure 3A; not significantly different by log-rank analysis, p>0.05). Thus, in the setting of adult lung epithelium MYC and K-rasG12D failed to cooperate to induce accelerated tumorigenesis.

Bottom Line: Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors.We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT

Background: Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as "oncogene-addiction." However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.

Methodology/principal findings: To examine how the MYC and K-ras(G12D) oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras(G12D) to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras(G12D)- or MYC/K-ras(G12D)-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras(G12D)-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras(G12D) resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras(G12D) in maintenance of lung tumors, we found that the down-stream mediators of K-ras(G12D) signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras(G12D) but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras(G12D). Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.

Conclusions/significance: Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.

Show MeSH
Related in: MedlinePlus