The systemic activin response to pancreatic cancer: implications for effective cancer cachexia therapy

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy partly due to frequent, severe cachexia. Serum activin correlates with cachexia and mortality, while exogenous activin causes cachexia in mice.

Methods: Isoform-specific activin expression and activities were queried in human and murine tumours and PDAC models. Activin inhibition was by administration of soluble activin type IIB receptor (ACVR2B/Fc) and by use of skeletal muscle specific dominant negative ACVR2B expressing transgenic mice. Feed-forward activin expression and muscle wasting activity were tested in vivo and in vitro on myotubes.

Results: Murine PDAC tumour-derived cell lines expressed activin-βA but not activin-βB. Cachexia severity increased with activin expression. Orthotopic PDAC tumours expressed activins, induced activin expression by distant organs, and produced elevated serum activins. Soluble factors from PDAC elicited activin because conditioned medium from PDAC cells induced activin expression, activation of p38 MAP kinase, and atrophy of myotubes. The activin trap ACVR2B/Fc reduced tumour growth, prevented weight loss and muscle wasting, and prolonged survival in mice with orthotopic tumours made from activin-low cell lines. ACVR2B/Fc also reduced cachexia in mice with activin-high tumours. Activin inhibition did not affect activin expression in organs. Hypermuscular mice expressing dominant negative ACVR2B in muscle were protected for weight loss but not mortality when implanted with orthotopic tumours. Human tumours displayed staining for activin, and expression of the gene encoding activin-βA (INHBA) correlated with mortality in patients with PDAC, while INHBB and other related factors did not.

Conclusions: Pancreatic adenocarcinoma tumours are a source of activin and elicit a systemic activin response in hosts. Human tumours express activins and related factors, while mortality correlates with tumour activin A expression. PDAC tumours also choreograph a systemic activin response that induces organ-specific and gene-specific expression of activin isoforms and muscle wasting. Systemic blockade of activin signalling could preserve muscle and prolong survival, while skeletal muscle-specific activin blockade was only protective for weight loss. Our findings suggest the potential and need for gene-specific and organ-specific interventions. Finally, development of more effective cancer cachexia therapy might require identifying agents that effectively and/or selectively inhibit autocrine vs. paracrine activin signalling.

Keywords: Activin receptor type IIb; Activins; Cachexia; Pancreatic cancer; Weight loss.

Figure 1

Murine PDAC tumour‐derived cells express activin A but not activin B, while tumours express both activins A and B. (A) Quantitative RT–PCR demonstrating varying levels of Inhba mRNA in cell lines: KPC32043, KPC32047, and KPC32908. Inhbb was undetectable (not shown). Data are presented as the mean fold change relative to normal mouse pancreas ± SD. (B) ELISA demonstrating activin A protein levels in the conditioned medium (CM) collected from the culture of the three KPC cell lines. Data are presented as mean ± SD. (C) Quantitative RT–PCR demonstrating levels of Inhba and Inhbb mRNA in pancreatic tumours generated by injection of the KPC cell lines into the pancreas of C57BL/6J mice. Triplicate replicate data are compared with normal pancreas of sham‐operated mice and presented as mean ± SEM (n = 3 tissues per condition). (D) Immunohistochemistry (IHC) of activin A or activin B in pancreatic tumours from two orthotopic PDAC models (KPC32043 and KPC32908). (E) IHC of pancreatic tumours from mice with genetic PDAC (KPC mice). Normal pancreas tissues were from sex‐matched and age‐matched mice with wild‐type (WT) Kras and WT p53 alleles. Scale bar: 100 μm. ^* P < 0.05, ^*** P < 0.001, ^**** P < 0.0001.

Figure 2

There is a systemic functional decline and organ wasting due to PDAC. (A–D) Systemic changes in body weight, activity, body condition, and organ weight in response to PDAC tumour developed by orthotopic injection of KPC32043 cancer cell line, compared with the sham‐operated mice. (E) Elevated activin A level in plasma from the tumour‐bearing mice as determined by ELISA. Data are presented as mean ± SEM. ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001.

Figure 3

There is a systemic activin response to PDAC. (A, B) Inhba or Inhbb mRNA expression by quantitative RT–PCR in organs harvested at the study endpoint from sham control and orthotopic KPC32043 tumour‐bearing mice. Expression values in all organs for each gene were normalized to the value from the control liver. IHC for activin A and activin B in organs from mice with (C) orthotopic KPC32043 tumours, (D) orthotopic KPC32908 tumours, or (E) genetic PDAC (KPC mice). Data are presented as mean ± SEM. Scale bar: 100 μm.

Figure 4

PDAC tumour cells express factors that induce muscle cell activin expression and atrophy. (A) Experimental scheme. (B) Quantitative RT–PCR demonstrating induction of Inhba and Inhbb by 75% KPC32908 CM. (C) Immunofluorescence staining for myosin heavy chain in C2C12 myotube cultures and quantification of myotube size (>200/condition) in growth medium GM, 25%, 50%, or 75% of KPC32908 CM. ^* P < 0.05, ^*** P < 0.001, ^**** P < 0.0001. Scale bar: 200 μm. (D) Western blot demonstrating activation of p38 MAPK by 75% CM from KPC32908 activin^high and KPC32043 activin^low at 15 and 30 min after CM treatment.

Figure 5

Activin blockade in mice with activin^low PDAC cells via soluble activin receptor reduces weight loss and prolongs survival. (A) Body weights after KPC32043 cell implantation into the pancreas and time points (arrows) of ACVR2B/Fc administration (10 mg/kg body weight via i.p.) (left) and body condition scores at the end of the experiment (right). IBW, initial body weight measured on the day of cell implantation. (B) Mouse activity measured at the indicated time points (left) or at the end of experiment (right) showing a therapeutic effect of activin blockade. (C) Increased survival in mice with KPC32043 tumours treated with ACVR2B/Fc (log–rank test). (D) Effect of activin blockade on organ mass. (E) Effect of activin blockade on the expression of atrogenes (left) or activins and related genes (right) in quadriceps. Data are presented as mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001.

Figure 6

Activin blockade in mice with activin^high PDAC cells via soluble activin receptor reduces weight loss without improving survival. (A) Body weights after KPC32908 cell implantation into the pancreas and time points (arrows) of ACVR2B/Fc administration (10 mg/kg body weight via i.p.) (left) and body condition scores at the end of the experiment (right). IBW, initial body weight measured on the day of cell implantation. (B) Survival in mice with KPC32908 tumours treated with ACVR2B/Fc (log–rank test). ^**** P < 0.0001.

Figure 7

Skeletal muscle‐specific blockade of activin signalling via expression of dnACVR2B in MLC‐dnACVR2B mice prevents PDAC‐induced weight loss but does not improve survival. (A) Body weights of KPC32043 pancreatic tumour‐bearing wild‐type (WT) C57BL/6 mice (WT + KPC32043) or transgenic (Tg) MLC‐dnACVR2B mice (Tg + KPC32043), normalized to each mouse's own initial body weight. (B) Body weights of KPC32908 pancreatic tumour‐bearing wild‐type (WT) C57BL/6 mice (WT + KPC32908) or transgenic (Tg) MLC‐dnACVR2B mice (Tg + KPC32908), normalized to each mouse's own initial body weight. (C) Survival of WT vs. Tg mice with KPC32908 tumour cells is not different (log–rank test). Mean ± SEM. ^* P < 0.05, ^** P < 0.01.

Figure 8

Activin proteins are expressed by human PDAC tumours. (A) Representative IHC for activin A and activin B in tumour tissues and quantification from PDAC patients from Indiana University (IUH). (B) Representative images and quantification of IHC for activin A and B in a PDAC cancer tissue microarray (TMA). Normal pancreas (n = 8) and PDAC tumour (n = 27). Mean ± SEM. Scale bar: 100 μm. ^**** P < 0.0001.

Figure 9

Survival of patients according to expression of activin family genes in pancreatic tumours. Gene expression vs. survival in the TCGA pancreatic adenocarcinoma data set. Ualcan.path.uab.edu accessed on 31 October 2018.

Figure 10

Proposed framework illustrating the systemic activin response to pancreatic cancer.