Periodic Reporting for period 1 - OPEN P-CAN (Dissecting the role of mitochondrial dynamics in pancreatic carcinogenesis)
Okres sprawozdawczy: 2020-04-01 do 2022-03-31
The proposal interrogated the role of mitochondrial dynamics in pancreatic cancer initiation and tested the hypothesis that oncogenic signaling drives changes in mitochondria to impact the nuclear epigenome. I had previously demonstrated that mitochondria-derived metabolites (i.e. acetyl-CoA) impact histone acetylation to facilitate pancreatic cancer onset. The research plan capitalized on the expertise of my mentor and project’s PI, Prof. Scorrano, whose work has pioneered the fields of mitochondria dynamics and inter-organelle communication.
Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest human cancers. Overall, 5-year survival is approximately 9%; nearly 80% of patients present with unresectable and/or metastatic disease. With limited therapeutic options for advanced disease, public health benefits stand to be gained by improving our understanding of the factors that contribute to tumor onset with the hope of curtailing disease incidence.
Mitochondria are intracellular organelles that serve critical bioenergetic and biosynthetic functions, but also influence nuclear chromatin. In particular, several studies indicate that histone acetylation is sensitive to mitochondrial activity. My central hypothesis was that mitochondrial structure is altered by KRAS signaling (aberrantly activated in PDA) and determines the relative risk for PDA.
Specifically, aims of the project were:
1) Characterize the link between mitochondrial function and histone acetylation in pre-neoplastic cells
2) Dissect mitochondrial dynamics (change in number, morphology) during PDA initiation
In brief, I found that that mitochondria exhibit tightening of inner membrane invaginations, termed cristae. This appear to be caused by upregulation of OPA1, a master regulator of cristae shape and biogenesis. Mouse models of OPA1 elevation show that cristae tightening is correlated with increased levels of histone acetylation and accelerated pancreatic carcinogenesis.
Understanding how mitochondrial and epigenetic reprogramming facilitate oncogenesis bear the potential to unveil unprecedented opportunities for the prevention of the disease. In particular, I identified tighter mitochondrial cristae in pre-malignant cells; targeting OPA1 (or cristae biogenesis in general) becomes an attractive strategy to block tumor initiation. The Scorrano laboratory has recently developed an OPA1-specific inhibitor that showed the ability to restrain pancreatic acinar cell plasticity ex vivo, which correlates with anti-tumorigenic potential. Its effectiveness in vivo will be tested.
Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest human cancers. Overall, 5-year survival is approximately 9%; nearly 80% of patients present with unresectable and/or metastatic disease. With limited therapeutic options for advanced disease, public health benefits stand to be gained by improving our understanding of the factors that contribute to tumor onset with the hope of curtailing disease incidence.
Mitochondria are intracellular organelles that serve critical bioenergetic and biosynthetic functions, but also influence nuclear chromatin. In particular, several studies indicate that histone acetylation is sensitive to mitochondrial activity. My central hypothesis was that mitochondrial structure is altered by KRAS signaling (aberrantly activated in PDA) and determines the relative risk for PDA.
Specifically, aims of the project were:
1) Characterize the link between mitochondrial function and histone acetylation in pre-neoplastic cells
2) Dissect mitochondrial dynamics (change in number, morphology) during PDA initiation
In brief, I found that that mitochondria exhibit tightening of inner membrane invaginations, termed cristae. This appear to be caused by upregulation of OPA1, a master regulator of cristae shape and biogenesis. Mouse models of OPA1 elevation show that cristae tightening is correlated with increased levels of histone acetylation and accelerated pancreatic carcinogenesis.
Understanding how mitochondrial and epigenetic reprogramming facilitate oncogenesis bear the potential to unveil unprecedented opportunities for the prevention of the disease. In particular, I identified tighter mitochondrial cristae in pre-malignant cells; targeting OPA1 (or cristae biogenesis in general) becomes an attractive strategy to block tumor initiation. The Scorrano laboratory has recently developed an OPA1-specific inhibitor that showed the ability to restrain pancreatic acinar cell plasticity ex vivo, which correlates with anti-tumorigenic potential. Its effectiveness in vivo will be tested.
I examined the status of the mitochondrial network in premalignant pancreata. KC mice, which express the oncogenic form of KRAS in the pancreatic epithelium, were sacrificed at 7 weeks of age – before the onset of neoplastic lesions. I documented mitochondrial structure by electron microscopy, measuring several morphometric parameters. Interestingly, mitochondria of KC mice exhibit a marked increase in cristae density, which is possibly mediated by OPA1. In line with this observation and consistent with the hypothesis, I found that OPA1 expression is upregulated upon mutant KRAS expression in multiple cell systems. This indicates that oncogenic KRAS reprograms mitochondrial dynamics.
To test the hypothesis that OPA1 mediates mutant KRAS-induced epigenetic reprogramming in pre-malignant cells, histone acetylation was assessed in OPA1-overexpressing (OPA1tg) acinar cells, both ex vivo (Western Blotting) and in vivo (IHC). In both cases, OPA1tg -derived acini showed significantly higher levels of histone acetylation, without significant changes in NAD+ levels. As elevated histone acetylation facilitates PDA initiation (Carrer, 2019), we anticipated that OPA1 overexpression accelerates tumor onset. To this aim, we bred OPA1tg with KC mice to obtain KC;OpaTg mice that showed higher histone acetylation, specifically in pre-malignant areas. Histological evaluation of 4-month-old animals exhibited the presence of advanced carcinomas only in OPA1-overexpressing animals.
Dissemination
Results were routinely discussed with the supervisor, both in one-on-one meetings and during lab meetings, to which the entire lab attended. The tools deployed for ex vivo analysis of isolated pancreatic acini were published in the form of a methodological review article (PMID: 32932616) in an open access, peer-reviewed, international scientific journal (Cancers, ISSN: 2072-6694). I was invited to present at a IUBMB-FEBS Joint Meeting in Sevilla (“Crosstalk Between Nucleus and Mitochondria in Human Disease”) and had the opportunity to engage with an international community broadly interested in inter-organelle communication. Unfortunately, most meetings were suspended in 2020-2021 because of the COVID pandemic so I had to forego several opportunities for networking and dissemination at the international level.
My profile and my research was highlighted by the University website and by the local press on multiple occasions, empowering the dissemination of my work to a broad and variegated audience.
To test the hypothesis that OPA1 mediates mutant KRAS-induced epigenetic reprogramming in pre-malignant cells, histone acetylation was assessed in OPA1-overexpressing (OPA1tg) acinar cells, both ex vivo (Western Blotting) and in vivo (IHC). In both cases, OPA1tg -derived acini showed significantly higher levels of histone acetylation, without significant changes in NAD+ levels. As elevated histone acetylation facilitates PDA initiation (Carrer, 2019), we anticipated that OPA1 overexpression accelerates tumor onset. To this aim, we bred OPA1tg with KC mice to obtain KC;OpaTg mice that showed higher histone acetylation, specifically in pre-malignant areas. Histological evaluation of 4-month-old animals exhibited the presence of advanced carcinomas only in OPA1-overexpressing animals.
Dissemination
Results were routinely discussed with the supervisor, both in one-on-one meetings and during lab meetings, to which the entire lab attended. The tools deployed for ex vivo analysis of isolated pancreatic acini were published in the form of a methodological review article (PMID: 32932616) in an open access, peer-reviewed, international scientific journal (Cancers, ISSN: 2072-6694). I was invited to present at a IUBMB-FEBS Joint Meeting in Sevilla (“Crosstalk Between Nucleus and Mitochondria in Human Disease”) and had the opportunity to engage with an international community broadly interested in inter-organelle communication. Unfortunately, most meetings were suspended in 2020-2021 because of the COVID pandemic so I had to forego several opportunities for networking and dissemination at the international level.
My profile and my research was highlighted by the University website and by the local press on multiple occasions, empowering the dissemination of my work to a broad and variegated audience.
The results I obtained identify dynamic changes of mitochondrial topology and structure during tumor-initiating metaplasia. This conclusion fetches a number of findings that represent significant scientific (and interdisciplinary) advancements:
• Mitochondria dynamics are extensively rewired by oncogenic signaling. I found that the mitochondrial network and structure are altered by mutant KRAS expression in that mitochondria are more fragmented and exhibit a tighter invaginations of their inner membrane.
• The epigenome senses changes in mitochondria shape.
• Mitochondrial shape influences tumor onset and progression. Overexpression of the cristae master regulator, OPA1 promotes pancreatic carcinogenesis in mouse models and its expression at high levels correlates with poor prognosis in humans.
All these findings are remarkably novel and represent a significant refinement of the state-of-the-art in the fields of mitochondrial dynamics and cancer epigenetics.
The project will move toward the characterization of the role of mitochondrial dynamics in determining the susceptibility to PDA. This will contribute to the identification of at-risk individuals, who will benefit of personalized prevention plans with the goal to detect the disease early because a lower stage at diagnosis correlates with better prognosis. We expect to define the transcriptional and epigenetic program associated with tumor initiation and the identification of novel biomarkers for early diagnosis. We also anticipate that compounds specifically targeting mitochondrial cristae biogenesis might be effective in suppressing disease progression at early stages. Combination with epigenetic drugs will also be evaluated.
PDA is a cumbersome disease, with abysmal survival rate and profound socio-economic impact as it represents a notable public health burden (currently the third-leading, projected to become the second-leading cause of cancer-related deaths by 2030). Economic impact is difficult to quantify, but average age at onset is ca. 50yo; this indicates that PDA hits individuals at the peak of their working age.
• Mitochondria dynamics are extensively rewired by oncogenic signaling. I found that the mitochondrial network and structure are altered by mutant KRAS expression in that mitochondria are more fragmented and exhibit a tighter invaginations of their inner membrane.
• The epigenome senses changes in mitochondria shape.
• Mitochondrial shape influences tumor onset and progression. Overexpression of the cristae master regulator, OPA1 promotes pancreatic carcinogenesis in mouse models and its expression at high levels correlates with poor prognosis in humans.
All these findings are remarkably novel and represent a significant refinement of the state-of-the-art in the fields of mitochondrial dynamics and cancer epigenetics.
The project will move toward the characterization of the role of mitochondrial dynamics in determining the susceptibility to PDA. This will contribute to the identification of at-risk individuals, who will benefit of personalized prevention plans with the goal to detect the disease early because a lower stage at diagnosis correlates with better prognosis. We expect to define the transcriptional and epigenetic program associated with tumor initiation and the identification of novel biomarkers for early diagnosis. We also anticipate that compounds specifically targeting mitochondrial cristae biogenesis might be effective in suppressing disease progression at early stages. Combination with epigenetic drugs will also be evaluated.
PDA is a cumbersome disease, with abysmal survival rate and profound socio-economic impact as it represents a notable public health burden (currently the third-leading, projected to become the second-leading cause of cancer-related deaths by 2030). Economic impact is difficult to quantify, but average age at onset is ca. 50yo; this indicates that PDA hits individuals at the peak of their working age.