Mice deficient for the gap junction protein Connexin32 exhibit increased radiation-induced tumorigenesis associated with elevated mitogen-activated protein kinase (p44/Erk1, p42/Erk2) activation
Timothy J. King1,2,3 and
Paul D. Lampe1,2
1 Cancer Prevention Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA and 2 Department of Pathobiology, University of Washington, Seattle, WA 98195, USA
3 To whom correspondence should be addressed Email: tking{at}fhcrc.org
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Abstract
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Loss of connexin expression/gap junction intercellular communication (GJIC) has been correlated with decreased growth control and increased tumorigenesis. Studies utilizing Connexin32 (Cx32)-deficient knockout mice have demonstrated that loss of Cx32 increases susceptibility to chemically induced liver tumorigenesis. Here, in addition to dramatically increased liver tumorigenesis, we show that tumor induction utilizing X-ray radiation resulted in a statistically significant increase in overall tumor burden in Cx32-deficient mice compared with wild-type mice due to tumorigenesis in several other tissues (lung, adrenal, lymph and small intestine) even when excluding prevalent liver tumors. Irradiated Cx32-deficient mice were particularly sensitive to liver tumorigenesis (46% incidence compared with 18% in wild-type mice, P = 0.007) demonstrating that Cx32 functions as a hepatic tumor suppressor in response to radiation-associated mutation events. Cx32-deficient mice also exhibited increased lung tumorigenesis (bronchioloalveolar) with an increased progression to carcinoma when compared with wild-type mice. Two Cx32-deficient mice developed an uncommon, invasive medullary adrenal tumor type (pheochromocytoma) not observed in irradiated wild-type mice. Immunohistochemical analysis revealed increased levels of activated mitogen-activated protein kinase (MAPK) (p44/Erk1, p42/Erk2) in Cx32-deficient mouse liver tumors (P = 0.006), lung tumors (P = 0.056) and adrenal tumors (primary and metastases) compared with wild-type counterparts implicating elevated activation of MAPK-interacting pathways in Cx32-deficient tumorigenesis. Interestingly, lung tumors from Cx32-deficient mice also demonstrated decreased p27Kip1 levels compared with wild-type lung tumors (P = 0.05). This study demonstrates that loss of Cx32/GJIC plays a significant role in radiation-induced tumorigenesis of the liver and importantly that Cx32 may also play a role in tumor suppression and/or tumor progression in other tissue types such as lung and adrenal gland. Additionally, this mouse model suggests that MAPK-related pathways may be preferentially activated or conversely that tumors harboring activated MAPK pathways may selectively progress towards more advanced tumor states in the absence of Cx32-mediated GJIC.
Abbreviations: Cx32, Connexin32; DEN, diethylnitrosamine; GJIC, gap junction intercellular communication
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Introduction
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Gap junction proteins, connexins, are a group of at least 20 highly conserved proteins that demonstrate developmental and tissue specific expression patterns (1,2). Gap junction intercellular communication (GJIC) allows for the direct transmission between neighboring cells of ions and small hydrophilic molecules <12 kDa in size; these include metabolites and messengers such as sodium, potassium, calcium, cAMP, ATP and inositol 1,4,5-triphosphate (1). Transmission occurs by passive diffusion through gap junction channels that span the plasma membranes of two adjacent cells. These channels are formed by the docking of two hemi-channels (connexons), each contributed by one of the adjoining cells; hemi-channels are comprised of a hexameric arrangement of connexins. GJIC/connexins play an important role in normal development and physiology and a loss of function is implicated in various human diseases including non-syndromic deafness, cataracts, inherited skin disorders and peripheral nerve disorders (3,4).
Several studies involving transgenic mouse strains with germline-inactivated connexin genes (knockouts) have revealed the importance of connexins in normal developmental and physiological processes (5) with animals demonstrating embryonic lethality (6,7) as well as abnormal phenotypes ranging from lethal cardiac malformations (8) to cataract formation (9) and disregulated liver function (10). Just as the presence of connexins, and presumably GJIC, have been implicated in the control of normal development and growth, the absence of GJIC in human and animal cells has been associated with decreased growth control and tumorigenesis (1,2). In several studies a lack of connexin expression or function was demonstrated in human tumors, namely prostate (11), liver (12), ovary (13), astrocytoma (14) and breast (15). It is unclear how much of the observed loss of connexin expression in tumor cells is a consequence of selective pressures during tumor growth in vivo or passage in cell culture in vitro. However, decreased Cx43 expression was reported in pre-neoplastic cells of the cervix (16). It can be hypothesized that the loss of growth-inhibitory signals from neighboring cells would aid in the progression of autonomous cell growth. Indeed, induction of GJIC between growth-inhibited normal and transformed cells (17) or exogenous expression of connexins in tumor cells restores at least partial growth control to the tumor cells in vitro and/or in vivo (1,2,16,18,19).
Particular attention has focused on mice genetically engineered to be deficient for Connexin32 (Cx32) (10). These mice exhibit disfunctional physiology including: abnormal bile secretion (20), altered pancreatic amylase secretion (21) as well as peripheral nerve demyelination reminiscent of human Charcot-Marie Tooth Syndrome (22). Interestingly, as Cx32 is the predominant mouse liver connexin, these knockout mice demonstrate altered liver function including decreased glucose mobilization possibly due to alteration of GJIC-mediated inositol triphosphate transfer (23). Most importantly, these mice exhibit increased susceptibility to chemically induced liver tumorigenesis observed as increased tumor frequency and tumor size following tumor induction with the liver-specific carcinogen, diethylnitrosamine (DEN) (2426). These mice also demonstrate abnormal synchronization of DNA synthesis following partial hepatectomy suggesting the possibility of altered cell cycle control in the absence of Cx32 (27). Restoration of Cx32 expression in immortalized and transformed liver cell lines resulted in decreased neoplastic potential (28,29).
However, in addition to liver, Cx32 is also expressed in a multitude of tissues including lung (30), kidney (31), intestine (32), pancreas (33), thyroid (34), testes (35) and ovary (36). To investigate the effect of Cx32 loss on tumorigenesis in many tissues, including liver, we exposed wild-type and Cx32- deficient mice to X-ray radiation and evaluated macro and microscopic tumor formation. Generally, Cx32-deficient mice exhibited a higher tumor burden compared with wild-type mice even when calculated excluding liver tumors. Knockout mice indeed exhibited increased liver tumor frequency and increased tumor size similar to results with DEN induction (2426). However, due to the use of radiation as a general tissue initiator, we also observed an increased sensitivity to lung tumorigenesis in Cx32-deficient mice. As published investigations into the molecular mechanisms underlying Cx32-related tumorigenesis are scarce, we chose to evaluate expression levels of several key targets of oncogenesis in these lung and liver tumors including: activated-mitogen-activated protein kinase (MAPK)/Erk1/Erk2, ß-catenin, Cyclin D1 and p27Kip1. Immunohistochemical analysis revealed an increased percentage of liver and lung tumors with elevated MAPK-activation (Erk1/Erk2) in Cx32-deficient mice compared with wild-type mice. In addition, Cx32-deficient mice uniquely exhibited MAPK-positive tumors of adrenal medullary tissue (pheochromocytoma) further implicating Cx32-loss with increased activation of MAPK-pathways and tumorigenesis.
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Materials and methods
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Mouse strains and radiation exposure
All mouse studies were conducted under FHCRC Institutional Animal Care and Use Committee approval. Cx32-deficient heterozygous mice originally created in the laboratory of K.Willecke (10) were generously provided by Steven Scherer (22) in a FVB/N background. These mice were crossed with C57-BL6 mice and inbred for five generations prior to the study. PCR analysis on DNA extracted from tail snips was conducted for mouse genotype determination as described previously (37). Wild-type mice (38 total, 14 male/24 female) and Cx32-deficient mice (29 total, 13 male/16 female) were irradiated at 14 ± 2 days of age with 4 grays (2.6 gray/min) total body exposure using a linear accelerator.
Necropsy, tissue processing, histological analysis
Mice were killed at first signs of morbidity, mortality or at 17 months of age. Necropsies were performed and all organs were macroscopically evaluated with subsequent formalin fixation of the following complete organs: liver, lung, pancreas, kidney, adrenal gland, intestine (duodenum), testes, ovary and any/all abnormal/tumorous tissues. All fixed tissue was embedded in paraffin and sectioned into 4 µm slices by standard procedures with subsequent de-paraffinization, hematoxylineosin staining and immunohistochemical detection. Pre-neoplastic foci were infrequent and due to their uncertain contribution to hepatocellular neoplasia were not classified or quantified. Liver tumor hepatocellular carcinoma classification was based on histological criteria including; mitotic figures, local tissue and blood/lymph vessel invasion, nuclear atypia, anaplastic appearance, trabecular morphology, increased nuclear:cytoplasmic ratio. Lung bronchioloalveolar carcinoma classification was based on criteria including: mitotic figures, local tissue and blood/lymph vessel invasion, nuclear atypia, anaplastic appearance, increased N:C ratio. Statistical analysis of tumor frequency was performed utilizing the Fisher Exact test. Volume calculations were performed utilizing the formula volume = (width2 x length/2) (19).
Immunohistochemical detection
Tumor slices were deparaffinized, blocked 1 h at room temperature in a 5% horse serum/PBS solution and detected utilizing a primary antibody against activated-MAPK (p44/Erk1, p42/Erk2) (1:100, Cell Signaling, Beverly, MA) or anti-tyrosine hydroxylase (1:5000, Calbiochem, La Jolla, CA) or anti-p27Kip1 (1:100, Neomarkers, Fremont, CA) in block, humidity chamber for 1 h at room temperature or overnight at 4°C (MAPK/Erk1/Erk2 antibody). Slides were then washed in PBS at room temperature three times for 5 min each followed by 1 h incubation with a biotinylated anti-rabbit (1:250, Vector Labs, Burlingame, CA) or anti-mouse (1:200, Southern Biotech., Birmingham, AL) secondary antibody for 1 h at room temperature. Following PBS washes, ABC-avidin/biotin conjugate (Vectastain, Vector Labs, Burlingame, CA) was applied for 30 min/room temperature with subsequent washes. Signal was detected with addition of diaminobenzidene (DAB) and counterstained with hematoxylin for nuclear detail. In cases where not all tumors were analyzed from both groups, a random representative selection of liver and lung tumors were subjected to immunohistochemical analysis. MAPK IHC reactivity was strong and quantified basically as binary data. Tumors with <2% positive cells were considered MAPK-negative tumors and tumors with >30% positive reactive cells were considered to be MAPK-activated tumors. In this study, no tumors were observed with intermediate percentages of reactive cells. p27 IHC results were quantified by counting the number of nuclei with strong p27 reactivity in two 40x microscopic fields for every lung tumor observed and presented as the average percentage of positive cells with range of field results. Nuclear signal was chosen over total cell or cytoplasmic signal, as use of this p27 antibody results in substantial non-specific cytoplasmic background.
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Results
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Cx32-deficient mice demonstrate increased susceptibility to radiation-induced tumorigenesis
Summary of radiation-induced tumorigenesis study. Cx32 is expressed in a multitude of mouse tissues including: liver, lung, kidney, pancreas, testes, thyroid, neural tissue, ovary and adrenal glands. Several studies have utilized a targeted-genetic knockout Cx32 mouse strain to evaluate the impact of Cx32/GJIC-deficiency on liver tumorigenesis employing tissue-specific chemical carcinogens (2426). We sought to evaluate the effect of Cx32-deficiency on radiation-induced tumorigenesis in a multitude of tissues including liver using this same Cx32-deficient mouse model. Utilizing X-ray radiation that is capable of initiating tumor formation in a wide spectrum of tissues, we exposed 38 wild-type mice and 29 Cx32-deficient mice to 4 grays of total body radiation with subsequent anatomical, histological and immunohistochemical analysis of liver, lung, kidney, pancreas, adrenal glands, testes, intestine, ovary and any other abnormal tissues at 17 months of age or upon detection of morbidity/mortality. Table I is an overall summary of tissue types exhibiting tumorigenesis presented as a frequency of specific tumor types (number of mice with particular tumor/total number of mice evaluated).
Cx32-deficient mice demonstrated a statistically increased tumor burden [animals bearing any tumor/total animals in group: wild-type, 14/38 (37%) tumor positive compared with Cx32-deficient, 20/29 (69%), P value = 0.007]. This comparison holds true even when excluding more prevalent liver tumors [wild-type 14/38 (37%), Cx32-deficient, 17/29 (59%), P value = 0.042]. There was no significant difference when comparing male with female mice in overall tumor susceptibility. While increased overall tumor burden is not indicative of increases in any particular tissue, these results suggest that Cx32-deficiency predisposes mice to increased tumorigenesis and sensitivity to radiation insult.
As listed in Table I, liver tumorigenesis was the most frequently observed tumor in Cx32 knockout mice and was statistically increased compared with wild-type mice (4618%, P value = 0.007) demonstrating that Cx32 acts as a tumor suppressor in liver following radiation-induced tumor initiation. While female mice demonstrated equal or slightly increased liver tumor frequencies compared with male mice, the average tumor volume was dramatically increased in male mice (Figure 1A) reinforcing the well-established sex bias in mouse liver tumorigenesis due to hormonal influence (38).

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Fig. 1. Cx32-deficient mice exhibit increased liver tumor volume. (A) Sex comparison of wild-type versus Cx32-deficient average liver tumor volume (mm3). Both = male + female, M = male, F = female. (B) Comparison of hepatocellular adenoma and hepatocellular carcinoma in wild-type and Cx32-deficient mouse liver. Error bars represent standard error for each group. All = adenoma + carcinoma; Ad = adenoma; Car = carcinoma.
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Lung tumor formation was also modestly increased in Cx32-deficient mice (2131% frequency) although this increase was not statistically significant. Additionally, Cx32-deficient mice demonstrated two occurrences of a less common medullary adrenal gland tumor type (pheochromocytoma). In both cases, these pheochromocytoma tumors were invasive with distant metastases detected in the lung.
Although Cx32 is expressed in the intestine (32), only a moderate, statistically insignificant, frequency increase was seen in intestinal adenoma formation suggesting that Cx32 expression alone is not critical for tumor suppression in this tissue. Additionally, it is noteworthy that several Cx32-expressing tissues (thyroid, pancreas, kidney, ovary and testes) were analyzed and exhibited no increased tumor sensitivity or obviously abnormal morphology implying that Cx32 may not play a unique tumor suppressive role in these particular tissues, at least in response to initiation by radiation. Several ovarian hyperplastic cysts were observed both in wild-type and Cx32-deficient mice but probably represent age-related and not Cx32-related etiology. It is unclear if there is any significance to the radiation-related thymic lymphomas observed in three Cx32-deficient male mice that lead to early morbidity (57 months). However, this tumor type was not detected in any wild-type mice and was excluded from specific tissue frequency totals. While histiocytic sarcoma incidence was elevated in Cx32-deficient mice (3/26), hemangiosarcoma incidence was increased in wild-type mice (3/38) complicating any direct conclusions from these observations.
Cx32-deficient mice display increased MAPK activation in radiation-induced liver tumors
All liver lobes from all animals were collected, processed, sectioned, stained and evaluated using histological techniques. Cx32-deficient mice demonstrated increased liver tumorigenesis compared with wild-type mice (Table I). The total number of tumors was significantly increased in Cx32-deficient mice (23 tumors/26 mice) compared with wild-type mice (9/38, P value <0.005). The average number of tumors/mouse (multiplicity) was only slightly increased in knockout mice (wild-type, 1.5/mouse; Cx32 KO, 1.75/mouse). Wild-type and Cx32 KO livers contained approximately equivalent ratios of hepatocellular adenomas and hepatocellular carcinomas indicating no dramatic adenoma:carcinoma bias in response to radiation (Table II).
However, the average tumor volume was dramatically increased in Cx32-deficient mice compared with wild-type mice (Figure 1A). Indeed, the majority of Cx32 knockout liver tumors were macroscopically evident compared with mostly microscopic wild-type liver tumors. Likewise, the average hepatocellular carcinoma volume was greatly increased compared with the average hepatocellular adenoma volume (Figure 1B). The adenoma/carcinoma percentage showed no sex bias in these mice. However, as mentioned earlier, tumor volume increased in males compared with females.
As increased activation of MAPK pathways are frequently observed in many tumor types including liver (39,40), we analyzed the majority of liver tumors detected in wild-type and Cx32-deficient mice immunohistochemically using an antibody reactive only against the phosphorylated/activated form of p44/p42-MAPK (Table II, Figure 2EH). Although immunohistochemical analysis is often not easily quantified, the antibody against activated-MAPK results in strong cellular signals allowing basically binary scoring capability. Specifically, tumors were determined to be negative if 2% or less of the cells in a tumor displayed significant IHC signals and were identified as positive if >30% of the cells within a tumor were labeled. In this study, no tumors displayed reactivity intermediate to these parameters resulting in a generally binary type of quantification.

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Fig. 2. Liver tumors from Cx32-deficient mice exhibit increased MAPK activation. Liver tumor sections stained with hematoxylin/eosin (left column, AD) and immunodetected for activated-MAPK/Erk1/Erk2 (right column, EH, brown immunoreactivity, blue hematoxylin nuclear counterstain. Photos depict the same specific tumor/same tumor region for each group photographed at the same magnification. (A and E) Wild-type hepatocellular adenoma. (B and F) Wild-type hepatocellular carcinoma. (C and G) Cx32 knockout hepatocellular adenoma. (D and H) Cx32 knockout hepatocellular carcinoma. Note mitotic figures in carcinomas (arrows, B and D). Bar represents 50 µM. See online supplementary material for color version of this figure.
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IHC analysis detected a significantly increased percentage of tumors positive for activated-MAPK in liver tumors from Cx32-deficient mice (Figure 2G and H) compared with wild-type liver tumors (Figure 2E and F). It is evident that MAPK activation is not strictly correlated with more advanced tumor classes (hepatocellular carcinoma) as MAPK-activated hepatocellular adenomas are also significantly increased in Cx32 knockout liver tumors compared with wild-type (Table II). As the only MAPK-positive wild-type tumor detected was a carcinoma, it is possible that normally MAPK-activation is indicative of tumor progression compared with Cx32-deficient liver tumors, which demonstrate MAPK-active adenomas. Cyclin D1 and ß-catenin levels are often dysregulated in liver tumors; we analyzed tumors for expression levels as well as localization of these proteins (not shown). IHC detection of ß-catenin generally demonstrated increased expression, but variability in localization and overall levels between individual tumors did not obviously correlate with tumor classification, genotype of animal or MAPK-activation. Additionally, when cyclin D1 levels were evaluated, the majority of tumors exhibited increased cytoplasmic and nuclear levels but, as with ß-catenin, did not seem to correlate generally with any other measured parameters. These results suggest that activation of these MAPK pathways in Cx32-deficient liver tumors is more prevalent than in wild-type tumors and that perhaps loss of Cx32 may increase the number of cells hyper-activating MAPK pathways or predispose initiated liver tumor cells to activate MAPK-related pathways resulting in tumor volume expansion. Further studies will be required to address and separate these two possible scenarios.
Cx32-deficient mice display increased MAPK activation in radiation-induced lung tumors
Histological analysis of all irradiated mouse lung tissue revealed a modest increase in lung tumor frequency in Cx32-deficient mice (31%) compared with wild-type mice (21%, Table I) although this was not statistically significant. The average number of tumors/mouse was slightly increased in Cx32-deficient mice (10/26 or 1 tumor/2.6 mice) compared with wild-type (8/38 or 1 tumor/4.8 mice, P = 0.072, Table III).
However, an increased percentage of total tumors were bronchioloalveolar carcinomas in Cx32 knockout mice (40%) compared with wild-type mice (12%, Table III). Additionally, while the significance of solid versus papillary bronchioloalveolar tumor morphology in the mouse is still undetermined, all tumors (adenoma and carcinoma) identified in the Cx32 knockout group exhibited papillary morphology in contrast with wild-type tumors that displayed only solid morphology suggesting that an unknown aspect of tumor progression differs between wild-type and Cx32-deficient mice (Figure 3AD) (P value <0.005).

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Fig. 3. Lung tumors from Cx32-deficient mice exhibit increased MAPK activation. Lung tumor sections stained with hematoxylin/eosin (left column, AD) and immunodetected for activated-MAPK (right column, EH, brown immunoreactivity, blue hematoxylin nuclear counterstain). Photos depict the same tumor/same tumor section for each group photographed at the same magnification. (A and E) Wild-type bronchioloalveolar adenoma (note small number of positive-staining infiltrating immune cells in E). (B and F) Wild-type bronchioloalveolar carcinoma. (C and G) Cx32 knockout bronchioloalveolar adenoma. (D and H) Cx32 knockout bronchioloalveolar carcinoma. Note solid morphology (A and B) compared with papillary morphology (C and D). Bar represents 50 µm.
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Mice irradiated at day 14 post-partum have lungs comprised predominantly of Cx32-expressing, multi-potent Type II pulmonary epithelial cells [up until approximately post-partum day 18 (41)]. Therefore, primarily Type II cells were subjected to tumor initiating damage. IHC analysis of Cx32 knockout lung tumors, utilizing a marker specific to normally Cx32-expressing Type II pulmonary epithelia (Surfactant C), confirms the Type II cell origin for these tumors (not shown). Altogether, these results indicate that loss of Cx32 may modestly increase lung tumor incidence, but more dramatically may allow increased progression of bronchioloalveolar adenomas to bronchioloalveolar carcinomas in mouse lung tumors.
Immunohistochemical analysis of lung tumors from irradiated mice (Table III) revealed an increased percentage of MAPK-activated lung tumors from Cx32-deficient mice (Figure 3G and H) compared with lung tumors from wild-type mice (Figure 3E and F, P = 0.056). The MAPK IHC staining in the lung was similar to that seen with liver in its binary quality. While activation of MAPK-related pathways appears to be correlated with tumor progression in mouse lung (4244), here it cannot completely explain the increased MAPK-activation in Cx32-deficient lung tumors as the sole bronchioloalveolar carcinoma detected in wild-type mice was completely negative for MAPK-activation (Figure 3F). However, as all four bronchioloalveolar carcinomas detected in Cx32-deficient mice displayed increased activated-MAPK levels compared with only one out of four bronchioloalveolar adenomas, the possibility that MAPK activation is associated with tumor progression must still be taken into consideration. These results suggest that loss of Cx32 may predispose lung tumors to increased progression toward carcinoma perhaps via increased activation of MAPK pathways. Furthermore, while increased activation of MAPK was also observed in liver tumors from Cx32-deficient mice, this differs from the above observations in one respect; in liver there was no increase in tumor progression (hepatocellular adenoma compared with hepatocellular carcinoma) whereas in lung there was an increase in progression.
As MAPK-activation leads to degradation of several cell cycle regulatory proteins, including the cyclin-dependent kinase inhibitor p27Kip1, whose loss is correlated with increased mouse lung tumorigenesis as well as increased tumor progression/invasion (45), we used IHC techniques to evaluate and quantify p27Kip1 levels in these same lung tumors. The number of tumor cells with a strong nuclear p27Kip1 IHC staining was quantified for every tumor observed and calculated as the number of positive cells/total cells/microscopic field. Interestingly, while nuclear p27Kip1 levels did not absolutely correlate with MAPK-activation in these tumors, all lung tumors (7/7) from wild-type mice (adenoma and carcinoma) demonstrated robust levels of immunoreactive nuclear p27Kip1 (7/7 tumors, mean 64 ± 5%, range 5369%, 1973 total cells scored; Figure 4A and B). In contrast, the lung tumors from Cx32-deficient mice fell into two groups: tumors with high p27Kip1 levels (4/8 tumors) demonstrating a mean percentage of 64 ± 5% of cells with strong nuclear p27Kip1 reactivity (range 5768%, 1695 total cells scored), essentially equivalent to tumors from wild-type mice, and lung tumors exhibiting lower p27Kip1 levels seen as fewer nuclear staining cells (4/8 tumors) with an average of only 6 ± 4% of cells with positive nuclear p27Kip1 reactivity (range 210%, 1411 total cells scored; Figure 4C and D) (P value = 0.05).

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Fig. 4. Lung tumors from Cx32-deficient mice exhibit decreased p27Kip1 levels. Lung tumor sections immunodetected for p27Kip1 (brown immunoreactivity, blue nuclear hematoxylin counterstain). (A) Wild-type bronchioloalveolar adenoma. (B) Wild-type bronchioloalveolar carcinoma. (C) Cx32 knockout bronchioloalveolar adenoma. (D) Cx32 knockout bronchioloalveolar carcinoma. Bar represents 50 µm.
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This decrease in p27Kip1 levels may result as a direct consequence of Cx32 loss or as a result of Cx32-associated tumor progression. Indeed, three out of the four total bronchioloalveolar carcinomas in the Cx32-deficient mice were in the group exhibiting decreased nuclear p27Kip1 levels. However, decreased p27Kip1 is not exclusively associated with carcinomas as one small adenoma demonstrated decreased p27Kip1 levels in contrast to a carcinoma from a wild-type mouse that contained high p27Kip1 levels. Additionally, the non-specific cytoplasmic background observed in these IHC experiments makes it difficult to ascertain any alteration in tumor-associated disregulation of p27Kip1 intracellular localization (46). Ultimately, these observed decreases in p27Kip1 levels may partially explain the increase in lung tumor progression from adenoma to carcinoma observed in Cx32-deficient mice.
Cx32-deficient mice exhibit MAPK-positive adrenal (pheochromocytoma) tumorigenesis
Two Cx32-deficient mice exhibited a less common medullary adrenal gland tumor type, pheochromocytoma. This type of tumor was not detected in any wild-type mice and is usually associated with mice genetically deficient for particular tumor suppressor genes such as neurofibromin (47,48). Both tumors observed were highly invasive with compression of cortical epithelial layers and disruption of the adrenal capsule ultimately manifesting in distant pulmonary metastases (Figure 5A and D).

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Fig. 5. Cx32-deficient mice exhibit MAPK-activated, invasive pheochromocytoma and lung metastases. Primary pheochromocytoma adrenal tumor sections (AC) and secondary lung metastases (DF). Tumor sections general stained with hematoxylin/eosin (A and D) and immunodetected for activated-MAPK (B and E; brown immunoreactivity, blue hematoxylin nuclear counterstain and immunodetected for tyrosine hydroxylase (C and F, no counterstain). C = adrenal cortex; PC = adrenal medullary pheochromocytoma tumor; L = lung; PC-Met = pheochromocytoma lung metastases. See online supplementary material for color version of this figure.
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These tumors were determined to be medullary in origin utilizing immunohistochemical detection directed against tyrosine hydroxylase, a medullary-specific enzyme involved in catecholamine synthesis (Figure 5C and F). Reactivity was detected only in the medullary portion of the primary tumors and not the cortical epithelia, more commonly the origin of mouse adrenal gland tumors (41) consistent with the observation that one wild-type mouse in this study developed an adrenal cortical adenoma with no detectable metastases in lung or any other analyzed tissue. Tyrosine hydroxylase- positive lung metastases were easily identifiable from immuno-negative surrounding pulmonary epithelia as well as immuno-negative tumors of lung origin. Interestingly, both primary tumors as well as secondary lung metastases were strongly positive for activated-MAPK further implicating MAPK pathways in Cx32-deficient tumor development and progression (Figure 5B and E). Additionally, analysis of hematoxylin/eosin stained sections with consecutive sections detected for MAPK-activation indicates that all secondary metastases were MAPK-positive. As there is a mixture of MAPK-positive and negative cells in the primary tumor (Figure 5B), MAPK-positive cells may represent more aggressive or more progressed cells within the primary tumor. The activation of MAPK pathways in these adrenal tumors correlates with the MAPK-activation detected in liver and lung tumors suggesting that loss of Cx32 may predispose mice to development of tumors in several different tissues perhaps as a result of increased activation of MAPK pathways or that Cx32-deficiency may preferentially allow for progression of tumors containing MAPK-activated pathways.
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Discussion
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Here, we demonstrate that Cx32-deficient mice are more sensitive to liver tumorigenesis following X-ray irradiation when compared with wild-type mice (Tables I and II, Figure 2). These results are similar to previous reports utilizing liver-specific carcinogens to evaluate Cx32 function in liver carcinogenesis (2426). However, we chose to employ radiation as the tumor initiator in order to evaluate the tumor modulating effect of Cx32 loss in many tissues that express Cx32 that are not targeted by this tissue-specific carcinogen. Radiation, while less efficient at initiating tumors in particular tissues than specific carcinogens, is capable of generating tumors in a multitude of Cx32-expressing tissues enabling a more general approach to evaluating the influence of Cx32 loss on tissue carcinogenesis. As a result of this approach, we demonstrate here that X-ray irradiated Cx32-deficient mice exhibit statistically significant increased tumor burden compared with irradiated wild-type mice (Table I) even when excluding the prevalent liver tumor burden. In addition, we demonstrate for the first time that irradiated Cx32-deficient mice exhibit increased lung tumorigenesis as well (Table II, Figure 3). It is of interest that radiation exposure did not generally increase tumorigenesis in other Cx32-expressing tissues such as pancreas, thyroid, kidney and intestine. While several tumors were observed in Cx32-deficient intestine, the frequency was quite low (7%) and equivalent tumors were also observed in wild-type animals. It is possible that Cx32 does not play a particularly important role in tumor suppression or cell proliferation in intestine, kidney, pancreas, etc., or that loss of Cx32-related tumor suppressor function can be compensated by redundant functions of other co-expressed connexin types.
Irradiated Cx32-deficient mice exhibited increased liver tumor volume as well as increased tumor frequency (Table II). Interestingly, these mice showed ratios of hepatocellular adenoma:carcinoma approximately equivalent to wild-type mice indicating that while loss of Cx32 does result in an increase in tumor volume, disruption of GJIC-mediated tumor suppression does not necessarily lead to accelerated tumor progression or increased carcinoma development following irradiation. Indeed, tumor volume increases may be partially explained by elevated hepatocyte proliferation observed previously in Cx32-deficient liver (24). Likewise, previously published studies have demonstrated increased pre-neoplastic foci and hepatocellular neoplasia in Cx32- deficient mouse liver in response to the tissue-specific carcinogen diethylnitrosoamine (DEN) (2426).
The loss of connexins and GJIC is highly correlated with decreased growth control and increased neoplastic potential both in vitro and in vivo (2), but the exact mechanism by which connexins exert tumor suppressive activity remains elusive. For example, studies have demonstrated that Cx32/GJIC is down-regulated in response to phenobarbital (49), a known liver tumor promoter, and that Cx32-deficient mice exhibit a lack of liver promotion following phenobarbital treatment (50). As radiation might be expected to cause tumorigenesis via a variety of mechanisms [in contrast to DEN initiation that has a more clearly directed oncogenic target Ha-ras (49)], we chose to investigate possible molecular mechanisms by which Cx32-deficiency might increase tumorigenesis. Here, we have demonstrated for the first time that liver tumors from irradiated Cx32-deficient mice exhibit increased levels of phosphorylated/activated-MAPK compared with tumors from wild-type mice (Table II, Figure 2). Increased MAPK activation has been observed in mouse and human tumors originating from many different tissue types with alterations of these particular pathways representing a very important mechanism of oncogenesis (40,51). Specifically, MAPK has been shown previously to directly modulate connexin/GJIC activity both in vitro and in vivo (52,53). Activated MAPK pathways have been correlated with aberrant connexin endocytosis (54). More pertinent to this study, decreased GJIC and increased MAPK activation has been demonstrated in rat liver epithelial cells following treatment with an immune modulator (55). Increased activation of MAPK pathways is particularly common in mouse liver tumorigenesis usually resulting from mutation of Ha-ras (49). While the microscopic properties of our tumors prevented PCR analysis in this study, it seems likely that tumors exhibiting increased MAPK activation would harbor Ha-ras mutations. It is interesting that studies utilizing DEN liver tumor induction showed decreased Ha-ras mutations and increased ß-catenin mutations with phenobarbital-related liver tumor promotion in wild-type mice (56). This is particularly attractive as connexin and ß-catenin pathways have been reported to interact at several distinct points of regulation (57,58). It is possible that DEN-induction, known to induce Ha-ras mutations in
30% of resulting liver tumors (49), affects alternate liver tumor promoting pathways in comparison with radiation-induced liver tumorigenesis. However, studies have shown that exposure to ionizing radiation does result in Ha-ras mutations in the liver (40% of tumors) suggesting that DEN and radiation may at least partially affect similar pathways necessary for liver tumor development (59). Regardless of exact mutations, Cx32-deficient mice exhibit an increased percentage of MAPK-activated liver tumors implicating this pathway in the increased susceptibility to liver tumorigenesis observed in this knockout mouse model. As equal percentages of hepatocellular adenomas and carcinomas exhibit activated-MAPK, loss of Cx32 most probably does not selectively allow development of MAPK-active carcinomas. It is possible that Cx32-deficiency either alters the capacity for initiated cells to acquire activation of MAPK pathways/ mutations and/or that initiated cells containing active-MAPK pathways are preferentially competent to expand and form detectable tumors.
In this study, we have for the first time microscopically investigated the possible role of Cx32 tumor suppression in various tissues in addition to liver in response to radiation exposure. We observed an increase in lung tumor frequency and progression in Cx32-deficient mice (increased percentages of broncioloalveolar carcinomas compared with adenomas) (Table III, Figure 3). This differs from liver in that Cx32 appears to play a more direct role in allowing increased tumor progression/conversion from lung adenomas to carcinomas whereas in liver this was not apparent. As in liver tumors, lung tumors from Cx32-deficient mice showed increased activation of MAPK pathways (Table III, Figure 3). This presumably is due to increased ras mutation (K-ras) within lung tumors. Indeed, K-ras mutations significantly increase in irradiated mouse lung (59). Although activation of MAPK pathways is often correlated with more advanced states of lung tumor (carcinomas) and while Cx32-deficient mice demonstrated increased percentages of carcinomas, MAPK activation is not necessarily solely a marker for tumor progression in these mice. Indeed, the sole carcinoma detected in wild-type mice was completely negative when evaluated for MAPK-activation. Therefore, the possibility remains that Cx32- deficiency results in not only an increased susceptibility to lung carcinogenesis but may also selectively allow tumors harboring activated-MAPK pathways/mutations to expand and progress toward carcinoma. Lung tissue expresses several different connexins that are developmentally as well as cell-type regulated (30,60,61). Type II epithelial cells (Cx32- positive) are multi-potent precursors to Type I epithelial cells (Cx43-positive) involved in gas exchange (41). At the time of irradiation in this study, neo-natal mouse pulmonary tracts are immature and underdeveloped, consisting primarily of Type II populated structures generally accepted as progenitors to the majority if not all of murine pulmonary neoplasia (41). Additionally, here, IHC staining of lung tumors using an antibody directed against Surfactant C (a Type II-specific molecule) demonstrated Type II tumor derivation (not shown). Therefore, the effect of Cx32 loss may be particularly significant in lung tumorigenesis as Type II cells also possess proliferative capacity. The effect of loss of other connexins normally expressed in Type II cells as well as the role of Cx43 (Type I cells) in lung tumor progression needs further evaluation (29,60,62).
Decreased p27Kip1 levels are associated with human and mouse lung tumorigenesis (45,63). Mice genetically deficient for p27Kip1 demonstrate increased susceptibility to chemical and radiation-induced lung tumorigenesis (45). Previous studies have detected increased p27Kip1 levels associated with increased cellular growth control following restoration of connexin expression/GJIC in lung and liver cells (29). Recently, Cx43 has been shown to modulate the degradation of Skp2 (an ubiquitin E3 ligase) resulting in altered p27Kip1 levels in cell culture (64). Interestingly in this study, all wild-type lung tumors (adenoma and carcinoma) expressed near normal p27Kip1 levels in contrast to half of the lung tumors from Cx32-deficient mice, which exhibited dramatically lowered levels of p27Kip1 (Figure 4). These results suggest that the Cx32-deficient lung may have decreased p27Kip1 levels predisposing tumors to increased growth and/or progression.
In further support of a link between Cx32-deficiency and increased activation of MAPK pathways in tumorigenesis, two Cx32-deficient mice exhibited invasive adrenal medullary tumors (pheochromocytoma) expressing activated-MAPK (Figure 5). These tumors were locally and distally invasive with detectable MAPK-positive metastases in the lung suggesting that this MAPK-activation is linked to increased tumor aggression. Although the frequency of pheochromocytoma in our study was low (2/29), this type of tumor is fairly uncommon and was not detected in any of the 38 wild-type mice. While the impact of connexin/GJIC loss in adrenal tumors of cortical origin has been established (65,66), the significance of Cx32 loss in tumors of medullary origin remains to be completely determined. Altogether, the increased tumor susceptibility demonstrated in these Cx32-deficient mice illustrates the important role that connexins and GJIC play as tumor suppressors/modulators.
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Supplementary material
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Supplementary material can be found at: http://www.carcin.oupjournals.org/
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Acknowledgments
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Special thanks to Christopher J.Kemp and Kay Gurley (Fred Hutchinson Cancer Research Center) for histological assistance, technical advice and various reagents. Thanks to Steven Scherer (Boston, MA) for providing Cx32-deficient mice. Thanks to Alix Joslyn, Ben Weigler, Shari Hunt, Jennifer Blackwood, Michael Golubev, Roberto Castaneda and the FHCRC Animal Health Resource Staff. This work was supported by NIH Grants AR47963, GM55632 (P.D.L.). T.J.K. was a recipient of an Institutional NIH-T32 Post-doctoral Research Training Grant (T32-AI07509).
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Received August 11, 2003;
revised November 24, 2003;
accepted December 19, 2003.