Category: Oncology Research

With over 2.2 million annual cases, breast cancer is the most common cancer in women, but drug resistance remains a formidable challenge, particularly due to the development of drug resistance in recurrent or metastatic cases.¹ The activation of the phosphatidylinositol 3-kinase (PI3K) pathway, driven by mutations in the PIK3CA gene, is a common oncogenic driver and plays a pivotal role in breast cancer development.²

Alpelisib, a PI3Kα-selective inhibitor, has shown promise in treating patients with PIK3CA-mutated breast cancer. However, studies have shown that certain mutations confer resistance to alpelisib, including upregulated retinoblastoma protein (Rb) and loss of phosphatase and tensin homologue protein (PTEN).³

Crucial Role of the piggyBac Transposon

In this context, a new study has harnessed the power of the piggyBac transposon system to unravel these key resistance mechanisms and propose a novel therapeutic strategy.

Auf der Maur and colleagues performed a genome-wide transposon-mediated mutagenesis screen using a mouse model of PIK3CAH1047R-driven mammary tumors.⁴ To do this, the team combined the piggyBac (PB) transposon system5 with a murine whey-acidic protein (WAP)-driven and PIK3CA^H1047R mutant mammary tumor model.⁶

The piggyBac system allowed for the identification of NF1 loss, a negative regulator of RAS, as a resistance mechanism against PI3Kα inhibitors. The study further elucidated the metabolic changes associated with NF1 loss, revealing enhanced glycolysis and lower levels of reactive oxygen species (ROS) in cancer cells.

By leveraging the piggyBac transposon system, this study has shed light on the mechanisms underlying resistance to PI3Kα inhibitors in breast cancer. The identification of NF1 loss as a resistance mechanism opens new avenues for more effective therapeutic strategies. As such, the group observed synergistic anti-tumor effects when NF1 knockout cells were treated with N-acetylcysteine (NAC) along with a PI3Kα inhibitor.

These findings also highlight the importance of utilizing innovative genetic tools like piggyBac to unravel resistance mechanisms and propel advancements in breast cancer treatment. This research offers hope for improved treatment strategies and underscores the power of the piggyBac system in advancing our understanding of drug resistance in breast cancer.

Learn More About piggyBac

The piggyBac system offers a versatile and efficient approach for genome editing, with applications across diverse research fields. Its remarkable cargo capacity of over 200kb sets it apart from other transposon and viral delivery vehicles, and makes it the ideal choice for creating stable cell lines.

To learn more about piggyBac and how Hera can help accelerate your gene editing research with our toolkit, click here.

References

1. Sung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A., Bray F. Global cancer statistics 2020: GLOBOCAN estimates of Incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer J. Clin. 2021; 71: 209-249. https://doi.org/10.3322/caac.21660
2. Miller T.W., Rexer B.N., Garrett J.T., Arteaga C.L. Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res. 2011; 13: 224 https://doi.org/10.1186/bcr3039
3. André F., Ciruelos E., Rubovszky G., Campone M., Loibl S., Rugo H.S., Iwata H., Conte P., Mayer I.A., Kaufman B. et al. Alpelisib for PIK3CA -mutated, hormone receptor–positive advanced breast cancer. N. Engl. J. Med. 2019; 380: 1929-1940 https://doi.org/10.1056/nejmoa1813904
4. Auf der Maur, P., et al. (Year). N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer. Cell Reports Medicine, 4(4), 101002.
5. Rad R., Rad L., Wang W., Cadinanos J., Vassiliou G., Rice S., Campos L.S., Yusa K., Banerjee R., Li M.A. et al. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice. Science. 2010; 330: 1104-110 https://doi.org/10.1126/science.1193004
6. Meyer D.S., Brinkhaus H., Müller U., Müller M., Cardiff R.D., Bentires-Alj M. Luminal expression of PIK3CA mutant H1047R in the mammary gland induces heterogeneous tumors. Cancer Res. 2011; 71: 4344-4351 https://doi.org/10.1158/0008-5472.CAN-10-3827

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The standard care of treatment for many types of cancer and infectious diseases around the world is currently chemotherapy. However, chemotherapies are widely known to be linked to severe toxicity and resistance that develop mostly due to non-specificity, leading to overall disease progression.

To combat these limitations, recent advancements in recombinant technologies have created some novel genetically engineered protein polymers such as Elastin-like polypeptide (ELP), Silk-like polypeptide (SLP), and hybrid protein polymers with specific sequences to impart consistent and precise features to target proteins, and which have given excellent preclinical results in studies so far.

The synthesis and administration of chemotherapeutics have thus made use of these protein polymers, overcoming many of the limitations of traditional therapeutics.

The research team responsible for a recent publication, summarizes the development of such advanced recombinant protein polymers, and discusses the main challenges associated with designing targeted delivery systems for chemotherapeutics, as well as the potential for unique gene editing platforms like Hera’s Cas-CLOVER to further enhance their applications in the future.

Challenges in traditional chemotherapeutics processing with recombinant protein polymers

Recombinant protein polymer research has grown significantly in popularity over the past few decades mainly because it has addressed a number of problems with traditional drug delivery systems. Many of these new polymers were discovered to be highly effective at delivering therapeutic payload, and some of them have even received FDA approval.

Natural protein polymers are utilized in forms such as nanoparticles, micelles, scaffolds, and hydrogels as the vehicles for targeted drug delivery to tissues.

See Figure 3:

a) Micelles
b) Hydrogel
c) Micelle containing targeting moiety and chemotherapeutics
d) Bi-functional micelle chemotherapeutics formulation
f) Modified liposomes with chemotherapeutics
g) Hybrid hydrogel synthesized by recombinant protein polymers and other protein polymers, like collagen, encapsulating chemotherapeutics.

Nevertheless, despite their effectiveness, there are a number of significant issues with these current systems that must be resolved.
Natural protein polymers are currently widely available, manageable, and affordable for use in a variety of biological applications, but they still have some drawbacks. For example, batch-to-batch deviations in raw materials can render the final products being prone to efficacy variance. Additionally, the molecular weight of the protein polymers limits their usage to only a few dozen applications at this time.

The future of recombinant protein polymers and targeted drug delivery systems

In response to these and other problems, genetic engineering has been used to develop recombinant protein polymers. These new polymers are synthesized using identical sequences and scaled up for large-scale production, thus resulting in protein polymers without as much batch-to-batch variance as before.

This technology offers new strategies for controlling molecular weight and the overall structure of the target protein. Additionally, other targeting sequences can be further fused with the protein for advanced drug delivery applications in the near future. The team in this study mentioned Cas-CLOVER as a great example for this application, as it has the potential to revolutionize the world of therapeutic bioprocessing by making precise, intentional and beneficial edits in the platform bioprocessing cell lines resulting in efficient and effective developmental control.

Accelerate your protein polymer formulations and bioprocessing projects with Hera’s Cas-CLOVER technology

Cas-CLOVER is a highly specific system demonstrating 99%+ efficiency, and it’s covered by issued patents with clear commercial access. To learn more about Cas-CLOVER, and how Hera can help accelerate your cell line development studies, click here. We have everything for your research needs, including flexible licensing options.

References:

1. Anjali Phour, Vidit Gaur, Ahana Banerjee, Jayanta Bhattacharyya. Recombinant protein polymers as carriers of chemotherapeutic agents. Advanced Drug Delivery Reviews, Volume 190 (2022), https://doi.org/10.1016/j.addr.2022.114544.

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Targeting KRAS In Oncology Research?

KRAS has become an important drug target in multiple cancers due to its central role in hypertrophic and pro-mitotic signaling. Hera provides several KRAS mutant xenograft cell line models in the SRG rat for researchers developing drug candidates targeting KRAS. KRAS is a small GTPase that is activated in response to growth factors including HGF, TGFα, and EGF. KRAS then activates the MAP kinase (MAPK; RAF/MEK/ERK) signaling pathway, driving ERK translocation to the nucleus, and resulting in transcription of pro-mitotic and pro-growth genes. Additionally, KRAS contributes to PI3K/AKT/mTOR signaling, leading to transcriptional activation as well as hypertrophic protein synthesis via mTOR¹ (figure 1).

Activating mutations in one of the three RAS genes (HRAS, NRAS, and KRAS) have been observed in up to 20% of all tumors, with mutations in the KRAS gene observed in ~90% of pancreatic cancers, ~50% of colorectal cancers, and ~25% non-small cell lung cancers (NSCLC)1. The G12D mutation is most common in pancreatic and the G12C mutation is most common in NSCLC, while many colon cancers harbor both the G12D and G13 KRAS mutations².

What Experimental Models Most Closely Replicate Human Tumors?

Heated interest in KRAS as an anticancer target has led to the development of numerous KRAS inhibitors such as Amgen’s Lumakras (sotorasib), recently approved for the treatment of NSCLC³. Although KRAS was first described in 1983, efforts to develop clinically effective KRAS inhibitors were largely unsuccessful until recently. Production of KRAS inhibitors has been stymied by lack of consistency in methods, reagents, and protein expression systems, with many groups using truncated KRAS proteins that exhibit altered tertiary structure and lack cell membrane interactions⁴. Furthermore, sensitivity to KRAS inhibition is highly cell-type specific, and numerous studies indicate that inhibition at multiple points in the KRAS signaling cascade may be necessary to achieve clinically relevant effects¹. Thus, patient-derived xenografts and stable cell lines expressing full length KRAS G12C, G12D, and G13 mutant proteins are required for preclinical evaluations of KRAS inhibition.

Hera BioLabs’ SRG OncoRat^® is immunodeficient and highly permissive to human xenografts and cell line inoculation. Developed on a Sprague-Dawley background, SRG OncoRats are Rag2/Il2rg double knockouts that lack mature B cells, T cells, and circulating NK cells⁵. Compared to immune-deficient mouse models, the SRG OncoRat^® provides the following advantages:

• Allows hosting for larger tumors (up to 10x volume compared to mice)
• Increased tumor take-rates
• Increased tumor growth kinetics
• Serial blood and tissue sampling for PK/PD studies
• Allows for the collection of efficacy data in the relevant toxicology/metabolism Sprague Dawley strain.

Which KRAS Mutant Cell Lines Have Been Successfully Grafted Into The SRG OncoRat^®?

To date, Hera BioLabs has validated four KRAS mutant lines in the SRG OncoRat^®: H358, HCT-116, MIA PaCa-2, and Capan-2 (table 1).

Capan-2, a human pancreatic ductal adenocarcinoma cell line, display epithelial morphology when grown in adherent tissue culture. Following xenograft into animals, Capan-2 form well-differentiated tumors. They express mutant KRAS (G12V) and elevated levels of the Epidermal Growth Factor Receptor (EGFR). In addition, they express wild-type p53 and normal levels of SMAD4 protein. When injected into immunocompromised SRG rats, they form well-differentiated tumors and are used as a PDX model for pancreatic cancer. The Capan-2 cells express mutant K-Ras (G12V), elevated Epidermal Growth Factor Receptor (EGFR), wild-type p53 and normal levels of the SMAD4 protein⁶. Figure 2 shows robust capan-2 tumor growth after inoculation into the SRG OncoRat^®.

H358 NSCLC cells contain cytoplasmic structures that are characteristic of Club cells. These cells only have the KRAS G12C mutation, without any other tumor suppressor or oncogenic mutations and they are exceptionally useful for the study of KRAS, EGFR, BRAF, MEK, and ERK signaling. Despite their KRAS mutation, H358 cells are sensitive to anti-GFR therapies. These cells were derived from a metastatic site⁷, and our preliminary data indicate that H358 cells form metastases in the SRG OncoRat®. Tumor growth in the SRG OncoRat^® is shown in Figure 3.

H441 Lung papillary adenocarcinoma cells were derived from a pericardial effusion metastasis in a 33-year-old man. In vitro, H441 form monolayers with epithelial barrier properties. These cells also harbor the TP53 A158L mutation and express surfactant protein A (SP-A). Like H358, H441 cells contain cytoplasmic structures that are characteristic of Club cells^7,8. H441 inoculation induces robust tumor growth in the SRG OncoRat^®, as shown in Figure 4.

HCT-116 human colon cancer cells display epithelial morphology and have been extensively characterized in proliferation, tumorigenicity, and drug screening studies. In vitro, HCT-116 are highly motile and subcutaneous xenografts in nude mice have demonstrated them to be highly tumorigenic⁹. When grafted into the SRG OncoRat^®, tumor take rate was 100% and tumor volumes reached 1800 to 12,000 mm³ by 24 days post-inoculation. When equal cell volumes were inoculated into NSG mice and SRG OncoRats^® tumors were roughly 5-fold larger in the rats (figure 5)⁵.

MIA PaCa-2 pancreatic ductal adenocarcinoma cells were obtained from a 65-year-old male. These cells are extremely well-characterized, with 1235 entries in PubMed. MIA PaCa-2 harbor TP53 mutations and homozygous deletions in the first 3 exons of CDKN2A/p16INK4A, without SMAD4/DPC4 mutations or microsatellite instability¹⁰. These cells express human colony stimulating factor, subclass I (CSF-I) and plasminogen activator. In vitro cells are adherent, with epithelial morphology. Subcutaneous inoculation with MIA PaCa-2 in the SRG OncoRat^® yielded consistent tumor growth (figure 6)⁵.

Targeting KRAS Mutant Positive Cancers With The SRG OncoRat®

The SRG OncoRat^® is a powerful tool for in vivo oncology studies, as it performs better than mouse models in several key ways. Our rats are extremely well-suited for hosting PDX and CDX cancer lines for analyses, including drug efficacy, tumor growth kinetics, and PK/PD studies. Tumor volumes are far larger than those obtained in mice, allowing for more detailed molecular characterization of tumors as well as PDX banking.

These qualities also make the SRG OncoRats^® attractive candidates for use as patient avatars, wherein
personalized precision therapies can be rapidly tested in vivo to guide clinical decision making.

If your preclinical studies could use a boost, or you would like to see more data on the SRG Rat, Contact Charles River Labs for more information.

References

1. Molina-Arcas, M., Samani, A. & Downward, J. Drugging the Undruggable: Advances on RAS Targeting in Cancer. Genes (Basel) 12, doi:10.3390/genes12060899 (2021).
2. Hobbs, G. A., Der, C. J. & Rossman, K. L. RAS isoforms and mutations in cancer at a glance. J Cell Sci 129, 1287-1292, doi:10.1242/jcs.182873 (2016).
3. Dunnett-Kane, V., Nicola, P., Blackhall, F. & Lindsay, C. Mechanisms of Resistance to KRAS(G12C) Inhibitors. Cancers (Basel) 13, doi:10.3390/cancers13010151 (2021).
4. Esposito, D., Stephen, A. G., Turbyville, T. J. & Holderfield, M. New weapons to penetrate the armor: Novel reagents and assays developed at the NCI RAS Initiative to enable discovery of RAS therapeutics. Semin Cancer Biol 54, 174-182, doi:10.1016/j.semcancer.2018.02.006 (2019).
5. Noto, F. K. et al. The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies. PLoS One 15, e0240169, doi:10.1371/journal.pone.0240169 (2020).
6. Kyriazis, A. A., Kyriazis, A. P., Sternberg, C. N., Sloane, N. H. & Loveless, J. D. Morphological, biological, biochemical, and karyotypic characteristics of human pancreatic ductal adenocarcinoma Capan-2 in tissue culture and the nude mouse. Cancer Res 46, 5810-5815 (1986).
7. Brower, M., Carney, D. N., Oie, H. K., Gazdar, A. F. & Minna, J. D. Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium. Cancer Res 46, 798-806 (1986).
8. Salomon, J. J. et al. The cell line NCl-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier. Mol Pharm 11, 995-1006, doi:10.1021/mp4006535 (2014).
9. Rajput, A. et al. Characterization of HCT116 human colon cancer cells in an orthotopic model. J Surg Res 147, 276-281, doi:10.1016/j.jss.2007.04.021 (2008).
10. Gradiz, R., Silva, H. C., Carvalho, L., Botelho, M. F. & Mota-Pinto, A. MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors. Sci Rep 6, 21648, doi:10.1038/srep21648 (2016).