Combination treatment with migrastatic inhibitors to target brain tumour spread

A Glioblastoma Multiforme (GBM), a malignant brain tumour, ranks highly among the most aggressive cancers. This study explored the effects of the antimigratory drugs, CCG-1423 and Rhosin hydrochloride, on the motility of cancerous cells. Scratch wound and tumour spheroid assays were conducted on untreated and treated U87 glioma cells to observe the extent of migration. The migratory cells from the assays were fixed with paraformaldehyde to assess the expression levels and distribution of actin and focal adhesions with confocal imaging. CCG-1423 and Rhosin hydrochloride used in combination were found to decrease the migration of tumour cells by targeting two different pathways. This was shown to be significant with p values of 0.004, 0.0098 and 0.0021. Treated cells showed different fluorescence levels of actin and focal adhesions in comparison to untreated cells. In the presence of the drugs, actin was more highly condensed within the cytoplasm rather than on the surface of the cells, and the focal adhesions became more pronounced with less co-localisation of the two. This research has paved the way for future studies, in which anti-migratory drugs have the potential to be used at the surgical site, as a complement to conventional treatment, to ultimately prevent the onset of secondary tumours.


Introduction
Glioblastoma Multiforme (GBM) is a form of malignant brain tumour (Figure 1), causing side effects ranging from personality changes to seizures, memory loss and mood disorders (Alexander & Cloughesy, 2017). Currently, only 5% of patients suffering from GBMs survive up to five years (Goodenberger & Jenkins, 2012) in comparison to melanoma and breast cancer at ~ 90% (Siegel et al., 2018), emphasizing the extent to which brain tumours are a national health issue. GBMs are relatively rare. The incidence of brain tumours over 2015-2017 was 12,071, in comparison to breast cancer, of which there were 55,176 new cases (CRUK, 2017). Therefore, less funding has been attributed to research in this area; Cancer Research UK (CRUK) spent as little as 2.7% of total funding on brain tumours in 2019/2020 (CRUK, 2020). In the temporal lobe, the formation of a lesion is faintly visible at grades 1 and 2.
(C,D) Ring-enhancing lesion with signs of necrosis and peritumoral edemas, as shown by the purple circles, indicative of a GBM (Alexander & Cloughesy, 2017). This is graded as stage 4, in which the tumour is highly infiltrative and heterogenous (Goodenberger & Jenkins, 2012).
Current GBM therapies involve surgical resection of the tumour, radiotherapy, and chemotherapy with temozolomide. An issue often encountered with surgery is the incomplete resection of the tumour, in which the cells left at the surgical site migrate and disseminate into other regions of the brain. These cells go undetected in magnetic resonance imaging (MRI) scans after surgery, leading to the formation of secondary tumours (Forst et al., 2014).
Ninety percent of cancer deaths are attributable to metastasis or recurrence in the case of high-grade tumours (Sun et al., 2015). This research provided a novel insight into the use of CCG-1423 and Rhosin hydrochloride as anti-migratory inhibitors for the purpose of overcoming long-term treatment failure and tumour recurrence. If placed at the surgical site via carmustine wafers, the motility of the remaining tumour cells could potentially be impeded until the patient can receive chemotherapy, typically two weeks after surgery has taken place.

Drugs Targeting Migration
The motility of tumour cells relies heavily on the contraction of actin, induced by myosin II. In some instances, myosin is activated by the guanosine triphosphatase   (Gandalovičová et al., 2017). Rac 1 induces the formation of new actin by activating the actin-related protein complex (Arp2/3) (Parri & Chiarugi, 2010). Rac1 and Cdc42 also activate Wiskott-Aldrich syndrome protein (WASP) and WASPfamily verprolin-homologous proteins (WAVEs). WASP and WAVE act as the nucleating promoting factors by binding to actin and the Arp2/3 complex. Adhesion proteins, specifically integrins, are relocated into the newly developed protrusions on the cancerous cells (Decaestecker et al., 2006), alongside focal adhesions, to enable communication between the tumour cells and the ECM (Tojkander et al., 2012). CCG-1423 targets a different cellular mechanism to Rhosin hydrochloride, in which it inhibits serum response factor (SRF) by binding to oncogene megakaryoblastic leukaemia transcriptional co-activator protein 1 (MKL1), both of which are upregulated in cancer ( Figure 3).

Figure 3 | Chemical structure of CCG-1423 (Tocris, n.d.) and mechanism of action.
Transcription of metastatic genes is induced by G-protein coupled receptor (GPCR) mediated RhoA activation, specifically by lysophosphatidic acid receptors (LPARs), and coupling to G-proteins, Gα12Q231L and Gα13Q226L (Vogt et al., 2003). Activation of RhoA induces actin polymerisation and the dissociation of MKL1 into the nucleus. In an unbound state, MKL1 activates SRF to initiate gene transcription. CCG-1423 acts by inhibiting SRF (Evelyn et al., 2007) and the localisation of MKL into the nucleus by binding to the nuclear localisation signal (NLS) region on MKL. This suppresses the binding of MKL1 to importin and MKL/SRF mediated gene transcription (Gau et al., 2017).
MKL and SRF are also responsible for the synthesis of cytoskeletal regulatory proteins to enable remodelling of the actin for cell migration (Gau et al., 2017). CCG-1423 prevents the interaction between MKL and SRF, supressing the transcription of genes inducing migration and metastasis (Biro et al., 2014). The drug also regulates MICAL2, a gene encoding F-actin monooxygenase, leading to the depolymerisation of actin (Gau et al., 2017). Without these pathways, the cells cannot migrate as the polarity of the cell ceases to change and myosin II remains inactive (Evelyn et al., 2007). The morphology of the migratory cancerous cells is also altered by CCG-1423, in which the protrusions are not as functional due to the depletion of ArpC2, vasodilatorstimulated phosphoprotein (VASP) and profilin 1, the actin-binding proteins involved in the formation of the cellular extensions (Gau et al., 2017).

Rhosin Hydrochloride
Previous studies showed the effects of drugs acting individually against various forms of cancer. Rhosin hydrochloride was previously studied on MCF7 breast cancer cell lines by Shang et al., where it was found to reduce the expression of RhoC and RhoA to suppress migration (Shang et al., 2012). Evelyn et al. reported that CCG-1423 inhibited invasion and migration in the PC-3 prostate cancer cell line by suppression of SRF and MKL1 gene transcription (Evelyn et al., 2007). This was the first scientific publication to introduce CCG-1423 as a potential anti-migratory drug. Although successful in other cancer cell lines, CCG-1423 proved ineffective in glioma cells and failed to inhibit migration as described by Ketchen et al. This paper proposed that a mesenchymal to amoeboid transition (MAT) occurred, in which the cancerous cells adapted their mode of migration to evade the effects of CCG-1423 (Ketchen et al., 2020). This highlighted the importance of using the drugs in combination to target both mechanisms. Rhosin hydrochloride would act to target amoeboid migration and CCG-1423 mesenchymal migration.

Cell Culture
The established glioma cell line U87

Scratch Wound Assays
The assay was performed following the protocol cited in a paper by Liang et al.,

2007.
Scratch wound assays were performed by seeding 1x10 " cells/ml in 6 well plates (Corning

ImageJ
Migration of the cells in the scratch wound assay was determined by ImageJ

Statistical Analysis
Results were analysed using Microsoft

Migration in a 2D Model
The    In combination, the migration index was significantly lower than that of the control (p = 0.00205).
The spheroids were imaged using the

Scratch Wound Confocal Analysis
The cells for both treated and untreated glioma cells appeared uniform. However, when treated with Rhosin hydrochloride, stress fibres were present ( Figure 14G). In combination, the cancerous cells formed clusters ( Figure 14H) with longer extensions, which are potentially filopodia.
Although protrusions aid migration, the binding of Rhosin hydrochloride to the GEF binding pocket suppresses actomyosin contractility, hence migration cannot occur.

Spheroid Confocal Analysis
Cells

Discussion
Cancer treatments are often less successful than predicted from pre-clinical studies due to the ability of cancer cells to adapt to adverse environments and challenges, such as the exposure to chemotherapeutic drugs.  forces (Harunaga & Yamada, 2011).

Conclusion
The mechanisms by which Rhosin hydrochloride and CCG-1423 work were already known in terms of their effect on intracellular events and molecules.
However, this study outlined their effects on cytoskeletal proteins as well, specifically focal adhesions and actin. This is a scientific breakthrough for brain tumour research as the current treatments are unsuccessful in prolonging the lives of patients with GBM for longer than 1.5 years, which is highly devastating. The drugs investigated here have the potential to stop migration at the site of surgical removal of the tumour to prevent tumour recurrence and formation of secondary tumours.