“Taken together, these data obtained using pancreatic cancer cells with KRAS mutations suggest the ability of the PCAIs to prevent metastasis and tumor growth, strongly indicating their potential to serve as effective targeted therapies for treating cancer types driven by the multiple mutant forms of KRAS.”
Pancreatic cancer remains one of the most difficult cancers to treat, in part because mutations in the KRAS gene are extraordinarily common in pancreatic ductal adenocarcinoma (PDAC). These mutations keep growth-promoting signals switched on, allowing cancer cells to proliferate and survive. Although drugs targeting certain KRAS mutations have emerged in recent years, they work against only a subset of mutant forms, leaving a need for strategies capable of targeting a broader range of KRAS-driven cancers.
A research paper published in Volume 17 of Oncotarget, titled “The anticancer effects of PCAIs in pancreatic cancer cells involve MAPK and PI3K/AKT pathways hyperactivation,” investigated a class of experimental compounds known as polyisoprenylated cysteinyl amide inhibitors, or PCAIs. Rather than simply shutting down signaling pathways normally associated with cancer growth, the researchers uncovered a more unexpected effect: PCAIs pushed some of these pathways into unusually high activity while simultaneously promoting oxidative stress, disrupting cell structure, and triggering cancer cell death.
The study was led by first author Kweku Ofosu-Asante, with Nazarius S. Lamango serving as corresponding author. Both are affiliated with the Florida A&M University College of Pharmacy Pharmaceutical Sciences, Institute of Public Health, in Tallahassee, Florida.
The Challenge of Targeting KRAS
KRAS belongs to the RAS family of proteins, which function as molecular switches controlling pathways involved in cell growth, differentiation, and survival. Mutations can interfere with the normal switching mechanism, leaving KRAS persistently active and promoting uncontrolled cell proliferation.
This problem is particularly important in pancreatic cancer, where KRAS mutations are extremely common. Current KRAS-targeted drugs such as sotorasib and adagrasib have represented an important therapeutic advance, but they primarily target the KRAS G12C variant. Other common mutations, including G12D and G12V, require different therapeutic approaches.
The researchers have been developing PCAIs as a potentially broader strategy. These molecules were designed to mimic structural features associated with the post-translational modification of RAS and other G-proteins. By interfering with polyisoprenyl-dependent protein interactions, PCAIs are intended to disrupt signaling processes used by oncogenic G-proteins rather than targeting only a single KRAS mutation.
Testing PCAIs in Pancreatic Cancer Cells
The researchers studied PCAIs primarily in two human pancreatic cancer-derived cell lines, PANC-1 and MIA PaCa-2, which carry different KRAS mutations.
They first compared 15 PCAI analogs to determine how effectively the compounds reduced PANC-1 cell viability. Two compounds, NSL-YHJ-2-45 and NSL-YHJ-2-27, emerged as the most potent, with EC50 values of approximately 3.6 and 3.8 micromolar, respectively.
Structural comparisons also provided clues about what made the compounds effective. PCAIs lacking the S-farnesyl group were largely ineffective, supporting the researchers’ hypothesis that this structural feature is important for disrupting the polyisoprenyl-dependent interactions of G-proteins.
Because NSL-YHJ-2-27 had previously shown strong activity against both PANC-1 and MIA PaCa-2 cells, it became the primary compound used for subsequent mechanistic experiments.
An Unexpected Response in Cancer Signaling
One of the most intriguing findings emerged when the researchers examined two major signaling networks downstream of KRAS: the MAPK and PI3K/AKT pathways.
Because these pathways normally promote cancer cell proliferation and survival, it might seem logical that an anticancer compound would work by suppressing them. The researchers initially expected something similar.
Instead, PCAI treatment produced the opposite response.
NSL-YHJ-2-27 increased phosphorylation of several components of the MAPK pathway. In PANC-1 cells treated with 5 micromolar of the compound, MEK1/2 phosphorylation increased by 129%, ERK1/2 by 150%, and p90RSK by 79%. Similar—and in some cases larger—changes occurred in MIA PaCa-2 cells, where ERK1/2 and p90RSK phosphorylation increased by approximately 270% and 250%, respectively.
AKT signaling was also strongly activated. After 48 hours of treatment with 5 micromolar NSL-YHJ-2-27, phosphorylation of AKT at Ser473 and Thr308 increased by 72% and 190%, respectively, in PANC-1 cells. MIA PaCa-2 cells showed increases of 97% and 82% at the same phosphorylation sites.
The finding presented an apparent paradox: pathways normally associated with cancer survival were becoming more active at the same time that cancer cells were dying.
When Too Much Growth Signaling Becomes Harmful
The researchers propose that the answer may lie in the degree of pathway activation.
Cancer cells depend on MAPK and PI3K/AKT signaling, but these pathways must remain within a range compatible with cellular survival. Previous studies have shown that excessive or prolonged activation of signaling proteins such as ERK and AKT can disrupt cellular homeostasis and promote cell death rather than continued proliferation.
In the PCAI-treated pancreatic cancer cells, MAPK and AKT hyperactivation occurred alongside several signs of apoptosis. The pro-apoptotic protein BAX increased, caspase-3/7 activity rose, and treated three-dimensional tumor spheroids showed extensive cell death and structural collapse.
The authors therefore suggest that PCAIs may push signaling beyond the level that cancer cells can tolerate. Rather than simply switching KRAS-associated pathways off, the compounds may disrupt their normal regulation so severely that signaling becomes detrimental to the cells themselves.
Oxidative Stress May Help Drive Cell Death
Another important clue came from measurements of reactive oxygen species (ROS).
Reactive oxygen species are chemically reactive molecules produced during normal cellular metabolism. At controlled levels, they participate in signaling, but excessive ROS can damage proteins, lipids, and DNA and contribute to cell death.
NSL-YHJ-2-27 caused substantial increases in ROS in both pancreatic cancer cell lines. At 3 micromolar, NSL-YHJ-2-27 increased ROS-associated fluorescence by approximately 240% in PANC-1 cells and 930% in MIA PaCa-2 cells compared with untreated cells.
The authors connect this response to the unusually strong MAPK and AKT activation. Previous research has shown that excessive signaling through these pathways can increase oxidative stress. In this model, PCAI-induced signaling hyperactivation and ROS accumulation may therefore form part of the process leading to apoptosis.
PCAIs Also Disrupted Proteins That Control Cell Movement
The effects were not limited to MAPK and AKT.
The researchers examined several small G-proteins involved in cellular organization and movement. Although PCAI treatment did not significantly alter KRAS protein levels, NSL-YHJ-2-27 substantially reduced RHOA and RAC1 in PANC-1 cells. At 5 micromolar, RHOA and RAC1 levels fell by 71% and 58%, respectively. RAC1 was also reduced in MIA PaCa-2 cells.
These proteins help regulate the actin cytoskeleton—the structural network that gives cells their shape and enables them to move.
Consistent with this effect, PCAI-treated pancreatic cancer cells lost prominent F-actin structures, became rounded, and occupied substantially smaller areas. At only 0.5 micromolar NSL-YHJ-2-27, mean cell area decreased by approximately 75% in PANC-1 cells and 65% in MIA PaCa-2 cells.
This disruption is particularly relevant to cancer because cytoskeletal remodeling and cell motility are essential components of invasion and metastasis.
Cancer Cell Migration and Invasion Fell Sharply
The researchers next tested whether these structural changes translated into reduced movement.
In wound-healing assays, NSL-YHJ-2-27 strongly inhibited migration in both pancreatic cancer cell lines. At 0.5 micromolar, migration was reduced by approximately 85% in PANC-1 cells and 92% in MIA PaCa-2 cells after 72 hours.
The researchers also examined three-dimensional spheroids embedded in Matrigel to model aspects of tumor invasion. At 10 micromolar NSL-YHJ-2-27, invasion was reduced by approximately 84% in PANC-1 spheroids and 96% in MIA PaCa-2 spheroids. At higher concentrations, the spheroids also began to disaggregate.
These experiments demonstrate strong effects on migration and invasion in vitro. They do not, however, establish that PCAIs prevent metastasis in animals or patients, which would require further studies in living systems.
Gene Expression Changed Across the Cancer Cells
To explore the effects more broadly, the researchers performed bulk RNA sequencing on MIA PaCa-2 cells treated with NSL-YHJ-2-27.
Initial analysis identified 201 affected genes. After applying the study’s statistical and fold-change thresholds, 88 genes were significantly differentially expressed: 85 were upregulated and three were downregulated.
Among the altered genes were several involved in oxidative stress, autophagy, signaling, and cancer-related processes. For example, HMOX1 and ATG9B were upregulated, while VCAN and SCHIP1 were downregulated. The researchers also validated increased SOCS1 protein expression following treatment.
These transcriptomic changes reinforce the idea that PCAIs do not act through a single isolated molecular switch. Instead, treatment appears to disturb multiple interconnected processes involved in signaling, cellular stress, structural organization, and survival.
Cancer Cells Underwent Apoptosis
The final pieces of evidence came from experiments directly examining cell death.
In three-dimensional PANC-1 spheroids, treatment with 5 and 10 micromolar NSL-YHJ-2-27 reduced the proportion of live cells by approximately 79% and 74%, respectively. Similar reductions of approximately 63% and 62% occurred in MIA PaCa-2 spheroids.
The spheroids also lost their compact structure and began to collapse at higher concentrations.
In PANC-1 cells, active caspase-3/7 levels increased substantially with PCAI treatment, while the pro-apoptotic protein BAX increased in both PANC-1 and MIA PaCa-2 cells.
Taken together, the results support apoptosis as an important component of the PCAI-induced loss of pancreatic cancer cell viability. The paper’s proposed model links this response to MAPK and PI3K/AKT hyperactivation, ROS generation, altered G-protein signaling, and disruption of the actin cytoskeleton.
Why These Findings Matter
Perhaps the most interesting aspect of the study is that it challenges the simple idea that anticancer therapy must always inhibit growth-promoting pathways.
MAPK and PI3K/AKT signaling normally helps KRAS-mutant pancreatic cancer cells grow and survive. Yet the findings suggest that disrupting the regulation of these pathways and driving them into excessive activity may also be harmful to cancer cells.
This concept could potentially offer another way of exploiting the biological dependence of KRAS-mutant cancers on tightly controlled signaling networks.
PCAIs may also have an advantage conceptually because they were designed to interfere with polyisoprenyl-dependent G-protein interactions rather than bind exclusively to one KRAS mutant form. The study therefore raises the possibility of developing agents with activity across cancers driven by different KRAS mutations.
That possibility remains preliminary. The experiments reported here were performed in pancreatic cancer cell lines and three-dimensional spheroid models. The study does not demonstrate that PCAIs are safe or effective treatments in humans, and the concentrations that produce anticancer effects in vitro cannot be assumed to be achievable or tolerable in patients.
Looking Ahead
The study provides mechanistic evidence that PCAIs can strongly disrupt KRAS-associated biology in pancreatic cancer cells. The compounds reduced cell viability, altered RHOA and RAC1 levels, disrupted the actin cytoskeleton, inhibited migration and three-dimensional invasion, increased oxidative stress, changed gene-expression patterns, and promoted apoptosis.
Unexpectedly, these anticancer effects occurred alongside hyperactivation rather than suppression of MAPK and PI3K/AKT signaling, suggesting that pushing normally growth-promoting pathways beyond a tolerable threshold may contribute to cancer cell death.
The authors conclude that the activity of PCAIs against pancreatic cancer cell lines carrying different KRAS mutations supports their potential applicability to multiple forms of KRAS-driven cancer.
However, substantial work remains before this approach can be considered a potential therapy. Future in vitro and in vivo studies will be needed to further assess PCAIs as potential pan-mutant KRAS-targeting agents and determine whether their anticancer activity can ultimately be translated safely into humans.
For now, the findings reveal an unusual vulnerability in KRAS-driven pancreatic cancer cells: pathways that normally sustain cancer growth may become damaging when their signaling is pushed too far.
Click here to read the full research paper published in Oncotarget.
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