Interaction between MYC and MCL1 in the genesis and outcome of non-small-cell lung cancer

Abstract

MYC exerts both positive and negative functions in cancer cells, such that its procancerous effects are unmasked only after its anticancer effects are blocked. Here we used multiple mouse models of lung adenocarcinoma to identify genetic events that can cooperate with MYC activation to promote the genesis of non-small-cell lung cancer (NSCLC), the most common form of lung cancer in humans. MYC overexpression targeted to pulmonary alveolar cells was sufficient to induce lung adenomas and carcinomas. Tumorigenesis was assisted by either spontaneous mutations in Kras or experimental introduction of activated RAS, but investigations revealed that additional events were required to circumvent apoptosis, one of the most significant negative functions exerted by MYC. We determined that overexpression of the antiapoptotic protein MCL1 was sufficient to circumvent apoptosis in this setting. Previous clinical studies have indicated that prognosis of human NSCLC is not associated with MCL1, despite its overexpression in many NSCLCs. In reexamining the prognostic value in this setting, we found that MCL1 overexpression does correlate with poor patient survival, but only when accompanied by MYC overexpression. Our findings therefore produce a convergence of mouse and human results that explain how MCL1 can block an important negative consequence of MYC overexpression in both experimental models and clinical cases of NSCLC.

Figure 1

Over-expression of MYC induced alveolar hyperplasia and apoptosis. (A) Schematic of transgenes used for MYC over-expression (see descriptions in text). (B) Analysis of transgenic MYC expression using whole lung RNA and a human-specific Taqman® probe. Each bar represents an individual mouse (n=3-4 each time-point; error bars not seen since deviation in individual mouse replicates was insubstantial). Values were normalized to the average background level of transcription detected in mice carrying the TM transgene alone (n=6 mice). (C) Western analysis of MYC levels in whole lung protein extracts. (D-F) H&E staining, (G-I) BrdU staining and (J-L) TUNEL staining of lungs of STM mice exposed to DOX for 0 (D, G, J), 4 (E, H, K) and 7 (F, I, L) days (bar in D equals 0.1mm; D-L same magnification).

Figure 2

Mice over-expressing MYC developed lung adenomas and adenocarcinomas. (A) Kaplan-Meier plot. (B) (B) Graphical representation of lung tumor incidence in transgenic mice. The cumulative numbers of tumors up until 18 months is displayed. (C-G, I, J) H&E stained sections from MYC transgenic mice. Adenomas (C, arrows) in TM mice remained small with well-circumscribed borders and a solid growth pattern (D). Large adenocarcinomas surrounded by smaller foci, presumably due to intra-pulmonary spread from the main tumor mass, could often be observed in DOX-fed STM (E) and CTM (F) mice. (G) Papillary patterning of an adenocarcinoma from a DOX-fed STM mouse. (H) Lung and trachea whole-mount. In this DOX-fed STM mouse, tumor tissue grew to occlude the entire left lobe of the lung before the mouse became moribund. (I) Adenocarcinoma invading the trachea of a CTM mouse. (J) Liver metastases with acinar histology from an STM mouse (bar in C, E, F, and I equals 1mm; bar in D, G and J equals 0.1mm).

Figure 3

MYC transgenic tumors have activating mutations in Kras. (A) Western analysis of MYC expression in normal lung, adenomas and adenocarcinomas from transgenic mice. (B) Spectrum of mutations detected in Kras from tumors from DOX-fed MYC transgenic mice (Green = wild-type, Red = mutant).

Figure 4

Activation of the MYC transgene inhibits tumorigenesis induced by activated RAS. (A) Kaplan-Meier plot. Mice were administered 50 mg/kg MNU intraperitoneally and monitored for up to 6 months. (B) Lesions visible on the pleural surface were quantified to compare tumor burden in MNU treated mice. Statistical significance was assessed using the Student’s T-test. (C) Schematic diagram outlining the protocol used to retrovirally transduce hyperplastic alveolar cells in STM mice. (D) Kaplan-Meier plot of mice transduced with pMig-HRAS^G12V. DOX treatment varied after transduction as indicated. (E) EGFP expression from an internal ribosomal entry site found in the pMig retroviral vector allowed tracking of transduced cells. Multiple EGFP⁺ adenomas and adenocarcinomas could be found 4-6 months after pMig-HRAS^G12V infection when mice were fed a DOX-free diet. (F) H&E stained lung section of a pMig-HRAS^G12V-infected mouse after 4 months on a DOX-free diet. (G) Frozen section showing an EGFP⁺ adenoma from a pMig-HRAS^G12V-infected mouse fed a regular diet for 1 month after transduction (blue DAPI counter-stain). (H) One month of DOX treatment was not sufficient to completely eliminate pMig-HRAS^G12V-transduced cells. EGFP⁺ cells could still be found in the alveolar region after one month of DOX treatment (arrows) (bar in E and F equals 1mm; bar in G and H equals 0.1mm).

Figure 5

MCL1 acts as an oncoprotein when co-expressed with MYC. (A) Of the anti-apoptotic BCL2 family proteins screened by Western analysis of lysates from lungs and tumors of DOX treated MYC transgenic mice (BCL2, BCL2L1/BCL-X_L, BCL2L2/BCLW, BCL2A1/BFL1, MCL1), only MCL1 was elevated in a subset of adenocarcinomas. Letters denote individual mice. (B) Retroviral constructs used to introduce Mcl1, Kras^G12V or both Kras^G12V and Mcl1 together into hyperplastic alveolar cells of STM mice. (C) Kaplan-Meier plot comparing the survival of mice fed a DOX diet following transduction with retrovirus encoding Kras^G12V alone or both Kras^G12V and Mcl1 together (Logrank, p<0.05). (D-F) H&E stained sections. (D) Lungs of an STM mouse transduced with Kras^G12V-expressing retrovirus and continuously fed a DOX diet to induce MYC over-expression. (E) Lungs of an STM mouse transduced with retrovirus co-expressing Kras^G12V and Mcl1 together and fed a DOX diet. (F) A tumor from a DOX-fed Kras^G12V/Mcl1 mouse showing large areas of both papillary adenocarcinoma (marked P) and large cell undifferentiated carcinoma (marked LC) in the same tumor mass. (G) Immunohistochemical staining for TTF-1 clearly demarcates the transition zone between papillary (TTF-1⁺) and large cell (TTF-1⁻) histology (bar in D and E equals 1mm; bar in F and G equal 0.1mm).

Figure 6

High MCL1 expression correlates with poor prognosis in human NSCLC co-expressing MYC. (A) Percentage of MYC positive and MYC negative human NSCLC that co-stained for high versus low levels of MCL1. MYC^pos tumors are those with an immunoreactive score (IRS) ≥ 1. MCL1^high tumors are those with an IRS > 4. (B) Kaplan-Meier plot of MYC negative NSCLCs dichotomized into MCL1^high (purple) and MCL1^low (blue) subsets. (C) Kaplan-Meier plot of MYC positive NSCLCs dichotomized into MCL1^high (purple) and MCL1^low (blue) subsets. p-values were estimated by Likelihood-ratio test.