Search

Supplemental materials include following: Supplemental Materials and Methods Table S1. NMII Concentration (n), nM Fig. S1. Model systems to human pancreatic cancer. Fig. S2. Scoring analysis and differential expression patterns of mechanoresponsive and non-mechanoresponsive paralogs in patient-derived IHC data. Fig. S3: Measurement of endogenous expression of nonmuscle myosin II paralogs and the effect of knockdown, overexpression, and 4-HAP treatment on the expression of mechanobiome proteins. Fig. S4. Pancreatic cancer cell lines are mechanically distinct from each other. Fig. S5: Method for quantifying actin structures at the cellular cortex. Fig. S6: 4-HAP decreases actin retrograde flow. Fig. S7: 4-HAP treated livers show reduced surface tumor coverage. Movie S1. Retrograde flow of an untreated AsPC-1 Sir-Act-stained cell. Movie S2. Retrograde flow of a 4-HAP-treated AsPC-1 Sir-Act-stained cell.

Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), α-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs—myosin IIB (MYH10), α-actinin 1, and filamin A—had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer.Significance:This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.

Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), α-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs—myosin IIB (MYH10), α-actinin 1, and filamin A—had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer. Significance: This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.

Metastatic disease is often characterized by altered cellular contractility and deformability, lending cells and groups of cells the flexibility to navigate through different microenvironments. This ability to change cell shape is driven in large part by the structural elements of the mechanobiome, which includes cytoskeletal proteins that sense and respond to mechanical stimuli. Here, we demonstrate that key mechanoresponsive proteins (those that accumulate in response to mechanical stress), specifically nonmuscle myosin IIA and IIC, α- actinin 4, and filamin B, are highly upregulated in pancreatic ductal adenocarcinoma cancer (PDAC) and in patient-derived pancreatic cancer cell lines. Their less responsive sister paralogs (myosin IIB, α-actinin 1, and filamin A) show a smaller dynamic range or disappear with PDAC progression. We demonstrate that these mechanoresponsive proteins directly impact cell mechanics using knockdown and overexpression cell lines. We further quantify the nonmuscle myosin II family members in patient-derived cell lines and identify a role for myosin IIC in the formation of transverse actin arcs in single cells and cortical actin belts in tissue spheroids. We harness the upregulation of myosin IIC and its impact of cytoskeletal architecture through the use of the mechanical modulator 4-hydroxyacetophenone (4-HAP), which increases myosin IIC assembly and stiffens cells. Here, 4-HAP decreases dissemination, induces cortical actin belts, and slows retrograde actin flow in spheroids. Finally, mice having undergone hemisplenectomies with PDAC cells and then treated with 4-HAP have a reduction in liver metastases. Thus, increasing the activity of these mechanoresponsive proteins (in this case, by increasing myosin IIC assembly) to overwhelm the ability of cells to polarize and invade may be an effective strategy to improve the five-year survival rate of pancreatic cancer patients, currently hovering around 6%. Citation Format: Alexandra Surcel, Eric S. Schiffhauer, Dustin G. Thomas, Qingfeng Zhu, Kathleen DiNapoli, Maik Herbig, Oliver Otto, Jochen Guck, Elizabeth Jaffee, Pablo A. Iglesias, Robert A. Anders, Douglas N. Robinson. Harnessing the adaptive potential of mechanoresponsive proteins to overwhelm pancreatic cancer dissemination and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3154.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality, with 37,000 people dying annually in the US. Existing strategies for treating cancer primarily mainly focus on inhibiting cell growth through specific genetic pathways, which typically either fail to completely abolish the disease or which lead to compensatory regulatory changes, and hence, drug resistance. Targeting cell mechanics remains an under-used approach for drug development. The direct driver of cell shape change events intrinsic to cellular functions, such migration and invasion, is the mechanobiome - a collection of cytoskeletal proteins which are the final determinants of a cell's mechanical attributes and which lie downstream of KRAS and other regulatory molecules. Targeting, and ultimately, inhibiting these processes is less likely to be subject to compensatory regulation by cancer cells. We determined via western blot analysis and immunohistochemistry of patient-derived samples that key players involved in mechanosensation-myosin IIA, IIC, α-actin-4, and filamin B -show increased expression in cancerous ductal epithelial over normal tissue, while non-mechanosensory, or variable mechanosensory, paralogs (myosin IIB, α-actin-1, and filamin A) show decreased expression. This upregulation of highly mechanosensory proteins has initiated an investigation into the necessity and sufficiency of the myosin II paralogs in PDAC metastasis through overexpression and knockdown of expression, coupled with mechanical assays. In addition to resolving the mechanobiome of PDAC, we have previously shown that targeting of myosin IIC by 4-hydroxyacetophenone affects PDAC mechanics. We are testing the in vivo efficacy of 4-HAP by conducting a murine multi-arm study of metastatically human derived pancreatic cancer cells. Preliminary results suggest a protective effect against the metastasis of human pancreatic cancer cells among mice treated with 4-HAP every other day. Citation Format: Alexandra Surcel, Qingfeng Zhu, Eric Schiffhauer, Robert A. Anders, Douglas N. Robinson. The mechanobiome of pancreatic ductal adenocarcinoma: a new, targetable drug space. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3811.

Cell engulfment mechanisms maintain metazoan tissue homeostasis by removing dying cells and pathogenic organisms. But recent evidence suggests that engulfment mechanisms also target viable cells and could therefore regulate rates of physiologic cell turnover in some contexts. One mechanism that targets live cells for engulfment is called entosis. By entosis, human tumor cells use the machinery of cell-cell adhesion to ingest their live neighbors, in a manner dependent on Rho-GTPase signaling and actomyosin. Live cells ingested by entosis are killed in a non-cell-autonomous manner by engulfing cells, following modification of entotic vacuoles by autophagy pathway proteins and lysosome fusion. We have shown that this mechanism of entotic cell death can limit transformed growth, suggesting a potential mechanism of tumor suppression linked to the engulfment and killing of live cells. Alternatively, we have shown that engulfing cells recover nutrients from ingested cells, and that entosis promotes the development of aneuploidy, suggesting that this process could promote tumor progression. Consistent with this, cell structures resembling those formed by entosis are observed most frequently in high-grade breast tumors that exhibit high rates of aneuploidy and aggressive clinical characteristics. Here we investigate the mechanism of entosis and consider that engulfments between neighboring viable cells could promote a form of cell competition, where “winner” cells engulf and kill neighboring “losers”. We find that differences in cortical mechanics (specifically, cortical deformability, which reflects the apparent elastic modulus and cortical tension) between neighboring cells, mediated by the Rho-GTPase pathway, dictate winner versus loser cell status, and that epithelial cadherin (E- and P-cadherin) expression is sufficient to induce this mode of competition between tumor cells. Tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that undergo cell death. These data define a mechanism of competition between mammalian cells that occurs in human tumors. Human tumors are known to be genetically heterogeneous, with many tumors exhibiting multiple independent clones identified by genetic alterations such as changes in ploidy. Entosis could allow neighboring cells to compete by acting as a mechanism of cell death toward losers, while selecting for more deformable winners and endowing them with ploidy changes and nutrient advantages that promote tumorigenesis. Our data add to an expanding list of mammalian cell competition mechanisms by demonstrating a mode of direct competition that utilizes cell engulfment, which is dependent on cell adhesion and differential cell mechanics, to allow winners to kill losers. Citation Format: Qiang Sun, Tianzhi Luo, Yixin Ren, Oliver Florey, Edmund S. Cibas, Louis Hodgson, Douglas N. Robinson, Michael Overholtzer. Competition between tumor cells by entosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY29-03. doi:10.1158/1538-7445.AM2014-SY29-03