The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models.Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration.Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals.Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation

Murine models for familial pancreatic cancer: Histopathology, latency and drug sensitivity among cancers of Palb2, Brca1 and Brca2 mutant mouse strains.

Alterations of the PALB2 tumor suppressor gene have been identified in familial breast, ovarian and pancreatic cancer cases. PALB2 cooperates with BRCA1/2 proteins through physical interaction in initiation of homologous recombination, in maintenance of genome integrity following DNA double-strand breaks. To determine if the role of PALB2 as a linker between BRCA1 and BRCA2 is critical for BRCA1/2-mediated tumor suppression, we generated Palb2 mouse pancreatic cancer models and compared tumor latencies, phenotypes and drug responses with previously generated Brca1/2 pancreatic cancer models. For development of Palb2 pancreatic cancer, we crossed conditional Palb2 null mouse with mice carrying the KrasG12D; p53R270H; Pdx1-Cre (KPC) constructs, and these animals were observed for pancreatic tumor development. Individual deletion of Palb2, Brca1 or Brca2 genes in pancreas per se using Pdx1-Cre was insufficient to cause tumors, but it reduced pancreata size. Concurrent expression of mutant KrasG12D and p53R270H, with tumor suppressor inactivated strains in Palb2-KPC, Brca1-KPC or Brca2-KPC, accelerated pancreatic ductal adenocarcinoma (PDAC) development. Moreover, most Brca1-KPC and some Palb2-KPC animals developed mucinous cystic neoplasms with PDAC, while Brca2-KPC and KPC animals did not. 26% of Palb2-KPC mice developed MCNs in pancreata, which resemble closely the Brca1 deficient tumors. However, the remaining 74% of Palb2-KPC animals developed PDACs without any cysts like Brca2 deficient tumors. In addition, the number of ADM lesions and immune cells infiltrations (CD3+ and F/480+) were significantly increased in Brca1-KPC tumors, but not in Brca2-KPC tumors. Interestingly, the level of ADM lesions and infiltration of CD3+ or F/480+ cells in Palb2-KPC tumors were intermediate between Brca1-KPC and Brca2-KPC tumors. As expected, disruption of Palb2 and Brca1/2 sensitized tumor cells to DNA damaging agents in vitro and in vivo. Altogether, Palb2-KPC PDAC exhibited features observed in both Brca1-KPC and Brca2-KPC tumors, which could be due to its role, as a linker between Brca1 and Brca2

Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele

<div><p>The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The <i>LSL-Kras</i>^{<i>G12D/+</i>};<i>LSL-p53</i>^{<i>R172H/+</i>};<i>Pdx-1-Cre</i> (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the <i>in vivo</i> investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new <i>Pdx1FlpO</i> knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the <i>Frt-STOP-Frt Kras</i>^<i>G12D</i> and <i>p53</i>^<i>Frt</i> mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent <i>in vivo</i> tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment.</p></div

<i>In vivo</i> Pdx1FlpO^ki allele expression specificity.

<p>(A) PCR analysis of Pdx1FlpO^ki mediated recombination of the p53Frt allele in the indicated tissues of <i>Pdx1FlpO</i>^<i>ki</i>;<i>p53</i>^<i>Frt/+</i> mice. (B) Representative GFP IHC staining demonstrates mosaic GFP expression in the pancreas and duodenum, but not the stomach, of <i>Pdx1FlpO</i>^<i>ki</i>;<i>FSF-GFP</i> mice. Scale bars = 25 μm.</p

p53 knockout accelerates PDAC formation.

<p>(A) Representative macroscopic view of pancreata from <i>Pdx1FlpO</i>^<i>ki</i>;<i>FSF-Kras</i>^<i>G12D</i>;<i>p53</i>^<i>Frt/+</i> mice at 3 and 6 months of age. (B) Representative microscopic H&E stained pancreatic sections from <i>Pdx1FlpO</i>^<i>ki</i>;<i>FSF-Kras</i>^<i>G12D</i>;<i>p53</i>^<i>Frt/+</i> mice at 3 and 6 months. Scale bars = 25 μm.</p

Generation of <i>Pdx1FlpO</i>^<i>ki</i> mice.

<p>(A) Schematic representation of the targeting vector, the wild-type Pdx1 locus, the Pdx1-FlpO/beta globin polyA/LoxP-Neo^R-LoxP targeting allele, and the Pdx1FlpO^ki allele after removal of LNL cassette. Mice containing the Pdx1FlpO^Pgk-NeoR allele were bred with Sox2-Cre-expressing transgenic mice to remove the LoxP-flanked NeoR cassette. Restriction sites: HindIII and AflII. Primer locations: p1, p2, p3, and p4. A Pgk-TX cassette was placed following exon 2 as a negative selectable marker. (B) Southen blot analysis: Genomic DNA from the aforementioned ES cells was digested by either HindIII or AflII, and hybridized with DNA probes that bind to either 5’ or 3’ of Pdx1 locus. (C) PCR analysis on both the 5’ (p1 and p3) and 3’ (p2 and p4) ends of the targeting vector, WT FVB/N mouse tail DNA, ES cell clone #3, ES cell clone #1, and tail DNA from the F0 chimera mouse generated from ES cell clone #3.</p

Metastasis spectrum in knock-in KPF mice.

<p>Metastasis spectrum in knock-in KPF mice.</p

Regulation of Epithelial Plasticity Determines Metastatic Organotropism in Pancreatic Cancer

The regulation of metastatic organotropismin pancreatic ductal a denocarcinoma (PDAC) remains poorly understood. We demonstrate, using multiple mouse models, that liver and lung metastatic organotropism is dependent upon p120catenin (p120ctn)-mediated epithelial identity. Mono-allelic p120ctn loss accelerates Kras(G12D) -driven pancreatic cancer formation and liver metastasis. Importantly, one p120ctn allele is sufficient for E-CADHERIN-mediated cell adhesion. By contrast, cells with bi-allelic p120ctn loss demonstrate marked lung organotropism; however, rescue with p120ctn isoform 1A restores liver metastasis. In a p120ctn-independent PDAC model, mosaic loss of E-CADHERIN expression reveals selective pressure for E-CADHERIN-positive liver metastasis and E-CADHERIN-negative lung metastasis. Furthermore, human PDAC and liver metastases support the premise that liver metastases exhibit predominantly epithelial characteristics. RNA-seq demonstrates differential induction of pathways associated with metastasis and epithelial-to-mesenchymal transition in p120ctn-deficient versus p120ctn-wild-type cells. Taken together, P120CTN and E-CADHERIN mediated epithelial plasticity is an addition to the conceptual framework underlying metastatic organotropism in pancreatic cancer

Kaplan-Meier survival with p53 inactivation.

<p>(A) Genetic strategy used to generate p53 heterozygous and homozygous deletion in <i>Pdx1FlpO</i>^<i>ki</i>;<i> FSF-Kras</i>^<i>G12D</i> mice. (B) Kaplan-Meier survival curves of the indicated genotypes. Median survival of <i>Pdx1FlpO</i>^<i>ki</i>;<i>FSF-Kras</i>^{<i>G12D/+</i>};<i>p53</i>^<i>frt/+</i> or <i>Pdx1FlpO</i>^<i>ki</i>;<i>FSF-Kras</i>^{<i>G12D/+</i>};<i>p53</i>^{<i>frt/frt</i>} mice is significantly lower than wild-type (WT) mice (p < 0.001, log-rank test for each pairwise combination).</p