The metabolic/pH sensor soluble adenylyl cyclase is a tumor suppressor protein

Abstract

cAMP signaling pathways can both stimulate and inhibit the development of cancer; however, the sources of cAMP important for tumorigenesis remain poorly understood. Soluble adenylyl cyclase (sAC) is a non-canonical, evolutionarily conserved, nutrient- and pH-sensing source of cAMP. sAC has been implicated in the metastatic potential of certain cancers, and it is differentially localized in human cancers as compared to benign tissues. We now show that sAC expression is reduced in many human cancers. Loss of sAC increases cellular transformation in vitro and malignant progression in vivo. These data identify the metabolic/pH sensor soluble adenylyl cyclase as a previously unappreciated tumor suppressor protein.

Keywords: cAMP; metabolic sensor; microdomain; sAC; tumor suppressor.

Figure 1. Expression of sAC mRNA is diminished in human cancers

(A–B) sAC differential mRNA expression in solid (A) and hematologic (B) human cancers compared to normal controls. Bar height represents the negative Log 10 of the P value of the comparison and the color indicates the fold change of sAC mRNA level relative to normal controls (blue colors indicate diminished expression and red colors indicate elevated expression). Data collected from Oncomine as described in the methods. SCC = squamous cell carcinoma. Embry. = embryonic. Carc. = carcinoma. Ter. = teratoma. YST. = yolk sac tumor. Sem. = seminoma. Blad. = bladder. BCC = basal cell carcinoma. PAC = prostate adenocarcinoma. Mel. = melanoma. AOA = anaplastic oligoastrocytoma. RCC = renal cell carcinoma. CRC = colorectal carcinoma. ACC = adenoidcystic carcinoma. Leu. = leukemia. Lym. = lymphoma. ALCL = anaplastic large cell lymphoma. GCBCL = germinal center B-Cell lymphoa. DBCL = diffuse B-Cell lymphoma. ABCL = activated B-Cell like.

Figure 2. Expression of sAC protein is diminished in human cancers

(A) Summary of immunohistochemical staining of normal human tissue (left bar) and human cancer (right bar) of data from the Human Protein Atlas using the anti human sAC antibody HPA017749. Color indicates staining intensity (white = no staining; light, medium, and dark blue = light, medium, and high staining intensity, respectively) and height of color is equivalent to the percentage of cases at that intensity. The number of cases analyzed in each group (N) is listed above the bar. (B) Representative example sAC expression (HPA017749, brown) in normal colon (left) and colorectal cancer (right) from Human Protein Atlas. In normal colon, sAC expression is mainly in the epithelial cells. (C) Representative example sAC expression (HPA017749, brown) in normal oral mucosa (left) and head and neck cancer (right) from Human Protein Atlas. In normal oral pharynx, sAC expression is mainly in the epithelial cells. (D) Normal human epidermis (left panel) and human squamous cell carcinoma of the genital skin (right panel) immunostained with anti-sAC antibody (R21; red chromagen). In normal skin, sAC expression is mainly in the epithelial cells. N = 10 (E) (Left panel) human cervical cancer (left arrow head) with adjoining normal cervix (right arrow head) and (right panel) basaloid vulvar cancer immunostained with anti-sAC antibody (R21; brown chromagen). Human tissue immunohistochemical staining was performed as previously described [25]. In normal cervix, sAC expression is mainly in the epithelial cells. N = 10 (D–E). In human skin staining there was 3+ staining in the epidermis (N = 6) and 1+ staining in squamous cell carcinoma (N = 6). In normal cervix there was 2+ staining and in cervical cancer (N = 10) there was 1 case with 0 staining of 100% of cells, 6 cases with 1+ staining of 100% of cells and 3 cases with 1+ staining of 90% of cells. In vulvar basaloid cancers (N = 6) all 6 cases showed 1+ staining of 100% of cells. SCC = squamous cell carcinoma. Ter. = Teratoma. YST = Yolk sac tumor. Sem. = Seminoma. Blad. = Bladder carcinoma. Gastric = Stomach adenocarcinoma. BCC = Basal cell carcinoma. PAC = Prostate adenocarcinoma. Mel. = Melanoma. AOA = Anaplastic oligoastrocytoma. RCC = Renal cell carcinoma. CRC = Colorectal carcinoma. ACC = Adenoid cystic carcinoma. Leu. = Leukemia. Lym. = Lymphoma. ALCL = Anaplastic large cell lymphoma. DBCL = Diffuse B cell lymphoma. ABCL = Activated B-Cell-Like. GCBCL = Germinal center B-Cell-Like. Co/Re = Colorectal. H/N = Head and Neck. Lymph. = Lymphoma.

Figure 3. Loss of sAC activity enhances transformation in vitro

(A–C) Cell growth of WT (squares) and sAC KO (circles) (A) 3T3, (B) SV40 and (C) HPV16-E6 MEFs in the absence (closed symbols, black lines) or presence (open symbols, red lines) of membrane permeable cAMP (B, 100 μM and C, 500 μM) for the number of days indicated. A-C, data presented is N = 4 for each data point. Experiments performed at least three times on two sets of independently produced cell lines. Legend below (A–C) identifies conditions. Notice: 3T3 wild type and sAC KO cells are contact inhibited. Repeated measures ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001. P Values indicated by brackets represent treatment group comparisons. (D) Colony formation (field image, upper panel) and quantitation (lower panel) of colony number of SV40 WT (WT, white bar) and sAC KO (KO, black bar) MEFs. N = 3 for each data point. Experiments performed at least three times on two sets of independently produced cell lines (E) Left panel, image of tumor formation in nude mice from WT (SV40 WT) and sAC KO (SV40 KO) after 60 days (arrow indicate tumors). Right panel, recorded tumor volume over time after injection with SV40 KO (5 million cells, red circles). SV40 WT (5 million cells, blue triangles) MEFs did not develop tumors. N = 10 for each genotype (F) Colony formation (field image, upper panel) and quantitation (lower panel) of colony number of E6 WT (WT, white bar) and sAC KO (KO, black bar) MEFs. N = 3 for each data point. Experiments performed at least three times on two sets of independently produced cell lines (G) Left panel, image of tumor formation in nude mice from WT (E6WT) and sAC KO (E6KO) after 30 days (arrows indicate tumors). Right panel, recorded tumor volume over time after injection with E6KO (red lines, 5 million cells [squares], 2 million cells [circles]). E6WT (black lines, 5 million cells, [triangles]) MEFs did not develop tumors. N = 10 for each genotype. Of note, while WT cells rarely formed colonies in soft agar, when WT colonies did form they were smaller on average than sAC KO colonies. Tumor xenografts were measured weekly using calipers, and a veterinary pathologist visually confirmed tumors after euthanasia. Error is represented as SEM. (D, F) Student's t-test, ***P < 0.001. F or E and G, experiment was reproduced using a distinct set of cell lines with identical results (data not shown).

Figure 4. Loss of sAC activity leads to MAPK pathway activation

(A–C) MAPK activity in mouse cells following serum starvation. Western blot and quantitation (normalized to WT) of pMEK/MEK (MEK Activity, 43 kDa, left panels) and pERK/ERK (ERK Activity, 44/42 kDa, right panels) in WT (white bars) and sAC KO (black bars) (A) 3T3 MEFs, (B) SV40 MEFs, and (C) E6 MEFs. For A-C, N = 3. (D) MAPK activity in 3T3 MEFs following the addition of Sp-8-CPT-cAMPs (+, 500 μM) or DMSO control (−) for 15 minutes. Data is a Western blot of pERK/ERK (44/42 kDa). Representative figure of an experiment performed in triplicate. Below Western blot is the average band quantitation (across all experiments) relative to DMSO control (set to 100%). (E) Western analysis of pERK (44/42 kDa) and total ERK (44/42 kDa) in WT (WT, top) and sAC KO (KO, bottom) MEFs following incubation for 1 hour with the MEK inhibitor GSK1120212 at the concentration indicated. Representative figure of an experiment performed three times. (F) Normalized cell number of WT (black lines, squares) and sAC KO (red lines, circles) MEFs following 72 hours of treatment with GSK1120212 at the concentrations indicated (N = 4). (G) Western blot and quantitation (normalized to WT) of pMEK/MEK (MEK Activity, 43 kDa, left panels) and pERK/ERK (ERK Activity, 44/42 kDa, right panels) in WT (white bars) and sAC KO (black bars) mouse epidermis. N = 3 (A–C, G) Student's t-test. error ± SEM. (F) Repeated measures ANOVA with post-hoc Sidak. Bracket indicates comparison of treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Loss of sAC enhances tumorigenesis in mice

(A) Image of tumor formation in wild type (left) and sAC KO (right) mice. N = 15 mice for both wild type and sAC KO cohorts. (B) Papilloma free survival in wild type (black line) and sAC KO (red line) mice. P = 0.0386. (C) Percentage of wild type (squares, black line) or sAC KO (circles, red line) mice with sustained papilloma growth greater than 2mm per lesion. (D) Papilloma formation per mouse in wild type (squares, black line) and sAC KO (circles, red line) mice. P = 0.0129. error ± SEM (E) Carcinoma-free survival in wild type (black line) and sAC KO (red line) mice. P = 0.0310. (F–H) Hematoxylin and eosin stained sections of representative examples of mouse skin lesions. (F) Example of normal mouse epidermis (inset image) in comparison to an example of focal thickening of the epidermis (papilloma, main image) due to inappropriate keratinocyte growth following DMBA/TPA treatment. (G–H) Squamous cell carcinomas from a sAC KO mouse at low power (G) and at a higher magnification to demonstrate invasion (H). (B, E) P values indicates the significant difference between the treatment groups following Kaplan-Meier survival analysis. (D) Repeated measures ANOVA. *'s refer to statistical difference at a given time point as determined by Sidak post-hoc analysis of the repeated measures ANOVA. *P < 0.05, and ***P < 0.001.